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the biogenesis of biological membranes that delineate the cell surface or act as intracellular space partition is a fundamental biological process and a remarkable evolutionary achievement . the hydrophobic barrier of membranes is comprised of fatty acid ( fa)-derived acyl - chains , typically 16 or 18 carbon atoms in length ; these fas are synthesized in a cyclic series of reactions by the multifunctional fa synthase ( fas ) complex by the addition of c2 units that are derived from malonyl - coenzyme a ( coa ) ( smith et al . , 2003 ) . remarkably , the fa chain length in membrane phospholipids ( pls ) is evolutionarily highly conserved ( daum et al . apparently provides maximum stability to the otherwise fluid bilayer structure through their hydrophobic interactions . at the same time , this acyl - chain composition permits rapid and significant adjustments by a single elongation step ( c16c18 ) and/or desaturation ( c16:0c16:1 ; c18:0c18:1 ) in response to alterations in environmental conditions such as temperature . furthermore , gradual increase in chain length and , subsequently , membrane thickness along the secretory pathway is an important determinant and provides sufficient differentiation for protein sorting ( bretscher and munro , 1993 ) . similar to mammalian cells , yeast membranes predominantly consist of mono- and di - unsaturated pls containing c16 and c18 acyl - chains to maintain the liquid crystalline state at physiological temperature ( de kroon et al . , 2013 ) . a decrease in the ambient temperature primarily triggers an increase in the c16/c18 ratio rather than a change in the degree of acyl - chain desaturation ( martin et al . , 2007 ) . the mechanisms that control the ratio between c16 versus c18 fas in cellular lipids are unknown . fas are synthesized de novo by the fas complex , which typically limits acyl - chain length extension to c16 and c18 carbon atoms , both in yeast and in mammals ( okuyama et al . , 1979 ; wakil et al . , 1983 ) ; fas are subsequently channeled through the common precursor phosphatidic acid ( pa ) into the synthesis of neutral and polar lipid species , i.e. , storage triacylglycerols ( tag ) and membrane pls , respectively . the substrate for the fas complex , malonyl - coa , is synthesized by the multifunctional enzyme , acetyl - coa carboxylase ( acc1 ) , the initial and rate - limiting step in cellular fa de novo synthesis in yeast ( al - feel et al . , 1992 ; chirala et al . , 1994 ; , 1993 ; woods et al . , 1994 ) and in mammals ( for a review , see wakil and abu - elheiga , 2009 ) . acc1 activity nutritional and metabolic signals , such as glucose limitation and salt stress , are transduced to acc1 by the snf1 kinase , the yeast ortholog of mammalian amp - activated protein kinase , ampk ( carling et al . , 1994 ; snf1 is the major energy - sensing kinase that phosphorylates and inactivates acc1 in vivo under conditions of low energy load ( woods et al . , 1994 ) . thus , yeast mutants lacking snf1 kinase display 3-fold elevated acc1 activity ; in addition , snf1 mutants are defective in transcription of multiple genes including ino1 , which encodes inositol-3-phosphate synthase and represents the most highly regulated gene of pl biosynthesis ( galdieri and vancura , 2012 ; jesch et al . , 2005 ; santiago and mamoun , 2003 ; shirra et al . , 2001 ) . inositol-3-phosphate synthase is indispensable for the endogenous production of inositol , which makes snf1 mutants dependent on inositol supplementation for growth ( ino phenotype ) . notably , transcription of acc1 and pl biosynthetic genes , including ino1 , is strongly regulated by inositol in the growth media , which is mediated by the uasino element in the promoter of these genes ( carman and han , 2009 ; henry et al . , 2012 , 2014 ) . full activation of ino1 transcription occurs in the absence of inositol and requires the ino2/ino4 transcriptional activator complex that binds to the uasino element ( ambroziak and henry , 1994 ; lopes and henry , 1991 ) . uasino - containing genes are repressed by opi1 ( white et al . , 1991 ) by its interaction with ino2 in the nucleus ( wagner et al . , 2001 ) . opi1 is a pa - binding protein and shuttles between the endoplasmic reticulum ( er ) and the nucleus , dependent on the level of pa and the presence of the yeast vamp - associated protein ( vap ) homolog scs2 , in the er . in the absence of inositol , pa levels are high , opi1 is bound to the er , and transcription of ino1 is enabled , leading to de novo production of inositol . in contrast , supplementation of inositol to the growth medium depletes pa by draining it into phosphatidylinositol ( pi ) synthesis , which promotes translocation of opi1 into the nucleus and repression of the ino1 gene ( loewen et al . , 2004 ; pa is a central lipid intermediate and precursor both for membrane pl and storage tag synthesis ; however , it is currently unclear how the metabolic flux either way is regulated in growing cells . defects in pl metabolism that lead to an increased steady - state concentration of pa in the er cause sequestration of the opi1 repressor to the er and elevated transcription of uasino - dependent genes . the cho2 and opi3 mutants , for instance , are defective in pl methylation and , thus , accumulate pa and overexpress ino1 leading to overproduction and excretion of inositol into the growth medium ( opi phenotype ; greenberg et al . , 1982 ; we previously showed that the inositol requirement of the snf1 mutant is suppressed by downregulation of acc1 , suggesting that its activity might be connected to the ino2/ino4/opi1 regulatory circuit ( shirra et al . however , due to the pleiotropic defects of the snf1 mutant , other regulatory mechanisms could not be ruled out . for instance , lack of ino1 expression in the snf1 mutant was suggested to be a result of defective chromatin modification ( lo et al . , 2001 ) . to uncouple acc1 activity from snf1-dependent phosphorylation , we have now characterized and mutated the specific phosphorylation site at position serine 1157 of acc1 . we provide evidence for a direct regulatory function of fa de novo synthesis in the transcriptional regulation of pl biosynthesis through the ino2/ino4/opi1 regulatory circuit . we show that acc1 activity is a critical determinant of the c16/c18 acyl - chain ratio in cellular lipids ; in conjunction with the specific binding preference of the opi1 repressor for c16-pa species , this describes a regulatory mechanism of gene transcription in response to altered acyl - chain length in cellular lipids and unveils a function of the snf1/amp - activated protein kinase in regulating lipid homeostasis . a large - scale phosphoproteome analysis identified a single phosphorylation site at serine 1157 of acc1 that is embedded in a mnravsvsdls sequence and matches the ampk consensus phosphorylation motif , r / k - x - x - s ( brinkworth et al . , 2006 ; dale et al . , 1995 ) . using avidin affinity - purified acc1 protein , we characterized and confirmed the phosphorylation site by qualitative and quantitative mass spectrometry analyses ( figure 1 ; figure s1 available online ; see supplemental experimental procedures for a detailed description ) . in wild - type , about 63% of the acc1 protein was phosphorylated at serine 1157 , suggesting that the majority of the enzyme is rather inactive in logarithmically growing yeast cells . however , in snf1 cells , the phosphorylation level at this residue in the acc1 protein decreased by half to about 35% , confirming serine 1157 as the target site of snf1 phosphorylation . alternative phosphorylation of serine 1159 , which was recently predicted to be a potential phosphorylation site ( oliveira et al . , 2012 ) , or phosphorylation of any other sites was not detected in our phosphoproteome analyses of log - phase yeast cells . to assess its in vivo function , we mutated serine 1157 to an alanine residue , thus locking the acc1 enzyme in a nonphosphorylatable state . as shown in figure 2a , the acc1 mutant , generated in this fashion , grew like wild - type on complete yeast extract peptone dextrose ( ypd ) media , but similarly to the snf1 mutant , it was resistant to the acc1-specific inhibitor , soraphen a ( sora ) ( shen et al . , 2004 ; vahlensieck et al . , in addition , acc1 mutants , like the snf1 mutant strain , were unable to grow on media lacking inositol ( i medium ) . this inositol auxotrophy ( ino phenotype ) could be suppressed either by decreasing acc1 activity with sora ( figure 2a ) or in the snf1 mutant by downregulation of acc1 transcription under control of the heterologous doxycycline - repressible tet07 promoter ( teto7-acc1 ; figure s2a ) . yeast snf1 mutants were originally identified by their inability to utilize exogenous sucrose ( snf , sucrose nonfermenting phenotype ) , resulting from their failure to express the suc2 gene ( encoding invertase ) on glucose depletion ( carlson et al . , 1981 ) . notably , the acc1 mutant was capable of growing on sucrose as the sole carbon source or on nonfermentable carbon sources like glycerol or ethanol , which distinguishes its phenotype from that of snf1 mutants with respect to carbon source utilization ( figures s2b and s2c ) . acc1 hyperactivity in both snf1 and acc1 mutants leads to highly elevated fa production and massive accumulation of tag and lipid droplets during logarithmic growth ( figures 2b and 2c ) ; these phenotypes are even more pronounced in stationary phase ( figure s2d ) . most notably , as a result of elevated malonyl - coa production by hyperactive acc1 in the acc1 mutant , fa acyl - chain distribution was significantly shifted toward longer acyl - chain lengths , increasing the c18:c16 acyl - chain ratio from 0.5 in wild - type to 2.0 in the acc1 mutant ( figure 2d ) . this shift is also reflected in the altered acyl - chain composition of virtually all glycerolipid classes for instance , tag ( figure s2e ) . taken together , the acc1 mutant displays similar phenotypes as the snf1 mutant with respect to fa overproduction , a shift to longer acyl - chains , and tag accumulation , as well as inositol auxotrophy . most notably , growth on nonfermentable carbon sources clearly distinguishes the acc1 mutant from the pleiotropic phenotypes that are associated with deletion of snf1 and uncouples acc1 activity from other roles of snf1 kinase in energy and carbon metabolism . the observed growth defect of acc1 mutants on solid media lacking inositol was reproduced in liquid cultures . when cultivated in the presence of inositol ( + i medium ) , the acc1 mutant grew at a rate identical to that of wild - type cells ( figure 3a ; figure s3a ) . after the shift to inositol - free medium , only a slightly reduced growth rate of the acc1 mutant strain was observed during the first 3 hr compared to wild - type , most likely due to intracellular inositol pools that are consumed during that period ( figure 3a ; see also figure s3b for detailed inositol titration experiment ) . however , a dramatic increase in doubling time of the acc1 mutant occurred between 3 and 6 hr after the shift to inositol - free medium ( figure 3a ) . notably , cells eventually reached wild - type - like final cell density after 20 hr , suggesting that cells are impaired in growth but remain viable ( figure s3a ) . this reduced growth of the acc1 mutant in the absence of inositol was cured by inhibition of acc1 by sora , clearly linking the requirement for inositol supplementation to acc1 activity . wild - type cells treated with the same concentration of sora , on the other hand , exhibited a severe growth defect in both the presence and absence of inositol , which is consistent with the essential role of acc1 in cellular metabolism ( figure 3a ) . we next asked whether the poor growth rate of the acc1 mutant in the absence of exogenous inositol was indeed associated with a reduced de novo production of that essential metabolite , i.e. , at the level of transcription of the ino1 gene . in wild - type , as expected , ino1 transcription was highly induced 3 hr following the shift to inositol - free medium ; in contrast , expression of ino1 was significantly attenuated in the acc1 mutant strain . consistent with the observed restoration of growth in the absence of inositol , inhibition of acc1 in the acc1 mutant by sora also restored wild - type - like ino1 expression kinetics ( figure 3b ) . these data support the notion that lack of growth of the acc1 ( and snf1 ) mutant on media lacking inositol is linked to a defect in ino1 expression as a consequence of acc1 hyperactivity . indeed , overexpression of ino1 from a high - copy - number plasmid or deletion of the opi1 gene fully restored growth of the acc1 strain in the absence of inositol ( figure 3c and figure s3c ) . in addition , downregulation of acc1 gene expression in the teto7acc1 strain by 0.53.0 g / ml doxycycline led to overproduction and secretion of inositol ( opi phenotype ; figure s4a ) , clearly demonstrating the inverse correlation between acc1 activity and the ability to express ino1 ( figure s4b ) . full repression of acc1 transcription ( > 5.0 g / ml doxycycline ) led to cell death of the teto7-acc1 strain , further supporting the essential function of acc1 . from previous studies , it is known that ino1 expression responds to the localization of opi1 , the transcription factor that senses the levels of the central lipid intermediate , pa , in the er ( loewen et al . , 2004 ) . consistent with the current model , opi1-green fluorescent protein ( gfp ) translocated in wild - type cells from the nucleus to the nuclear and peripheral er within 3 hr in inositol - free medium , leading to the observed rapid ino1 induction . in marked contrast , the acc1 mutant retained nuclear localization of opi1-gfp after the shift to inositol - free medium ( figure 3d ) ; this defect in opi1-gfp export from the nucleus parallels the reduced ino1 expression kinetics . notably , inhibition of acc1 activity in the acc1 mutant readily promoted export of opi1-gfp from the nucleus , leading to the observed relief of ino1 expression ( figures 3b and 3d ) . taken together , these analyses demonstrate that this growth deficit of the acc1 mutant is due to impaired ino1 expression through the ino2/ino4/opi1 regulatory circuit . since opi1 localization responds to the amount of pa in the er ( loewen et al . , 2004 ) , we hypothesized that overproduction of fa might lead to reduced cellular pa levels , perhaps due to its increased channeling into tag synthesis . this , however , was not the case : analysis of the lipid profile in the acc1 mutant showed that total glycerophospholipid content remained almost unaltered compared to wild - type . diacylglycerol ( dag ) and tag levels were raised about 3- to 4-fold , which was reversed by sora treatment within 4.5 hr ( figure 4 ) . thus , elevated de novo synthesized fas are preferentially channeled into tag synthesis , without affecting total pl content . we next analyzed to what extent cellular pa content was altered as a result of hyperactive acc1 in the acc1 mutant . surprisingly , the total pa pool was not decreased but rather slightly increased compared to wild - type ( figure 5 ) . , that reduced pa levels are responsible for translocation of opi1 from the er into the nucleus to repress ino2/ino4-dependent ino1 transcription ( loewen et al . , 2004 ) . since the molecular species distribution in all glycerolipids displayed a characteristic shift toward increased acyl - chain length in acc1 , we hypothesized that not the total amount of pa but rather its specific molecular acyl - chain composition may define its binding affinity to opi1 . thus , we next analyzed the molecular species composition of pa ( and of the major cellular pl phosphatidylcholine [ pc ] as a reference ) in wild - type and acc1 strains in greater detail . as shown in figure 5 , pa 32:1 ( containing c16:0 and c16:1 acyl - chains ) and pa 32:2 ( containing two c16:1 acyl - chains ) species were indeed greatly reduced in the acc1 mutant , whereas pa 36:1 ( containing c18:0 and c18:1 acyl - chains ) and pa 36:2 ( containing two c18:1 acyl - chains ) species were increased . this shift in acyl - chain composition was reversed by the addition of sora , a condition that also led to inositol prototrophy ( figures 2a and 3a ) , derepression of ino1 ( figure 3b ) , and nuclear export of opi1-gfp ( figure 3d ) in the acc1 strain . based on the hypothesis that an increase in the proportion of c18 versus c16 acyl - chains in pa is responsible for nuclear localization of opi1 , we next tested whether exogenous c18:1 supply and concomitant changes in the acyl - chain composition would phenocopy the effects of the hyperactive acc1 allele also in wild - type cells . as shown in figure 6a , addition of c18:1 indeed induced inositol auxotrophy in wild - type cells . this phenotype was not observed in the presence of palmitoleic acid ( c16:1 ) and , thus , can not be due to a general lipotoxic effect of supplied fa on inositol - free media . similarly , the ole1 mutant , which lacks the sole fa desaturase in yeast ( stukey et al . , 1989 ) , and which is dependent on unsaturated fa supplementation , displayed a strict inositol auxotrophy in the presence of c18:1 . notably , growth of wild - type , ole1 , and acc1 strains on media lacking inositol was fully rescued by ( co)supplementation with c16:1 , indicating that this fa reversed the inositol auxotrophy induced by excess c18:1 fa . this rescue of the acc1 mutant growth was not due to repression of acc1 activity by c16:1 , since total c18 fas remained at a high level , characteristic for acc1 hyperactivity ; rather , the ratio between c16 and c18 fas was restored by c16:1 supplementation ( figure 6b ) . the analysis of pa species in wild - type and ole1 strains supplemented with c18:1 showed a similar shift toward longer acyl - chain lengths as in the acc1 strain as compared to wild - type grown without c18:1 ( figure 6c ) : pa 32:1 and pa 32:2 species were significantly decreased , whereas pa species with longer fa chain length increased , especially 36:2 . to further confirm whether the acyl - chain composition of pa affects the interaction with opi1 , we performed in vitro binding assays with pc 36:2 liposomes as a matrix , containing 20 mol% of different pa species . indeed and in line with our hypothesis , we found significantly different binding affinities of opi1 for pa species dependent on the acyl - chain composition . liposomes containing saturated pa species ( pa 32:0 and pa 36:0 ) showed the highest affinity , followed by liposomes containing 32:1 pa ; liposomes containing pa 36:2 showed the lowest binding affinity for purified opi1 . liposomes , which consisted solely of pc 36:2 , showed no opi1 binding at all ( figure 7 ) . these results clearly demonstrate that the acyl - chain composition of pa is a major determinant of its interaction with the transcriptional repressor opi1 . this preferred binding affinity explains the observed phenotypes and significantly extends the current model of the transcriptional regulation of pl biosynthesis in yeast in response to deregulated fa de novo synthesis . throughout the eukaryotic kingdom , fas of 16 or 18 carbon atoms in length are the preferred acyl components in all glycerolipids that serve as bulk lipids in biological membranes or as storage lipids . whereas mechanisms controlling the level of fa desaturation in response to ambient temperature and membrane fluidity have been described in detail , much less is known about the mechanisms regulating chain length distribution ( de kroon et al . , 2013 ) . in addition to their fundamental roles as components of biological membranes , fas also serve essential functions as energy substrates , signaling molecules , or protein modifiers . cellular fa synthesis is also coordinated with nutritional supply , activation , and incorporation into complex lipids to support proper cell function and to prevent potentially detrimental fa - induced lipotoxic effects , which are believed to be causative for lipid - associated disorders , such as obesity and type 2 diabetes in humans ( kim and ye , 2013 ; unger , 2002 ; vidal - puig and unger , 2010 ) . thus , endogenous de novo synthesis of particular fas also plays an important role to counterbalance the physiological impact of nutritional fas that may be incorporated into cellular lipids . according to their central role in cellular lipid synthesis , both acc1 and fas have emerged as potential drug targets in obesity - related diseases , as well as in cancer , which are characterized by deregulated fa and lipid metabolism ( wakil and abu - elheiga , 2009 ; furuta et al . , 2010 ; pandey et al . , 2012 ; wang et al . , 2010 ; liu et al . , 2010 ; natter and kohlwein , 2013 ) . in yeast and mammalian cells , acyl - chain length is largely determined by the fas complex that limits acyl - chain length to 16 and 18 carbon atoms ( okuyama et al . , 1979 ) . we now show that the activity of acc1 , the initial and rate - limiting enzyme of de novo fa synthesis , is a major determinant of total cellular fa production and also has a fundamental impact on the relative distribution of c16 and c18 acyl - chains in cellular lipids . overproduction of fas leads to the accumulation of tag and proliferation of cytosolic lipid droplets , whereas membrane pl content remains unaltered . on the other hand , the shift in acyl - chains toward longer residues is reflected in all cellular glycerolipids , with consequences for the binding and sequestration of the transcriptional repressor opi1 to the er . the synthesis of fas is a major energy- and carbon - consuming pathway and , therefore , under several levels of control . one level involves the expression of the acc1 gene , which is coordinately regulated with pl synthesis by the pl precursors , inositol and choline , and the positive and negative transcription factors ino2/ino4 and opi1 , respectively ( hasslacher et al . , 1993 ; henry et al . , the regulation of acc1 expression in coordination with pl biosynthetic genes ( for review , see tehlivets et al . , 2007 ) suggests that fa production must also be balanced with tag formation in growing cells in order to sustain requirements for pl and membrane biogenesis . in addition , the activity of acc1 is strongly regulated by snf1-dependent phosphorylation ( mitchelhill et al . , 1994 ; woods et al . , 1994 ) , which , in turn , depends on the energy status of the cell ( wilson et al . , 1996 ) . here , we made use of a yeast mutant that lacks the single snf1 phosphorylation site in acc1 at serine 1157 ( acc1 ) and exhibits phenotypes very similar to snf1 mutants with respect to tag and lipid droplet accumulation as well as inositol auxotrophy , demonstrating that these snf1 phenotypes are caused by acc1 hyperactivity . the evidence we present here strongly supports the hypothesis that the inositol - requiring phenotype of the acc1 strain is due to decreased ino1 expression that is controlled by the well - characterized ino2/ino4/opi1regulatory circuit ( henry et al . , 2012 ; loewen et al . , 2004 ) , as both deletion of opi1 , which uncouples transcriptional repression of ino1 from cellular pa levels , and overexpression of ino1 fully restored inositol prototrophy to the acc1 mutant strain . we further suggest that pl synthesis may be limited by the coordinated repression of the uasino - containing pl biosynthetic genes in the presence of excess fa . the observed opi1 translocation to the nucleus concomitantly slows cell growth as pl and inositol production become limiting due to pa diversion into tag accumulation . most notably , neither increased endogenous fa production nor exogenous fa supply led to increased pl accumulation , despite the fact that an altered acyl - chain composition is reflected in all pl classes ; instead , excess fas are preferentially channeled into tag . in addition , the similarity of the cellular response to these two situations suggests that functional tag synthesis is crucial for accommodating these fas , thus avoiding the buildup of potential signal molecules , such as pa or dag , or free fas ( garbarino and sturley , 2009 ; kohlwein , 2010 ; kohlwein and petschnigg , 2007 ; petschnigg et al . , 2009 ; fakas et al . , importantly , our evidence significantly extends the current model that indeed the acyl - chain composition of pa is the critical determinant of opi1 sequestration to the er . we show that alterations in the acyl - chain composition of the acc1 strain or of wild - type and the ole1 mutant in the presence of oleic acid lead to inositol auxotrophy due to the reduced binding affinity of the opi1 repressor to pa molecular species containing longer acyl residues . we hypothesize that the recognition of the negatively charged head group of pa by the basic tract of opi1 might only represent the first level of interaction , whereas the second level is presumably determined by sensing the nature of the acyl - chains of pa within the er membrane . several other studies also support the importance of acyl - chain composition of pa for interaction with proteins ( kooijman and burger , 2009 ) ; for example , the binding specificity of human protein phosphatase 1 ( pp1c ) to pa species harboring a higher degree of acyl - chain unsaturation ( jones and hannun , 2002 ) . however , no consensus sequence motif for pa binding proteins has been identified yet ( stace and ktistakis , 2006 ) . it should be noted that the acidic pls pa and pi are exceptional regarding their membrane activity compared to other pls with respect to impact on membrane packing , electrostatics , and membrane curvature ( bigay and antonny , 2012 ) . pa is the only anionic pl with a pronounced cone shape , facilitating protein penetration into the membrane bilayer and recognition of the acyl moieties ( kooijman et al . . the actual concentration of pa in the yeast er membrane is not well defined . previous studies reported pa levels between 0.5% and 3% of total pls in isolated microsomal fractions ( zinser et al . , 1991 ; pichler et al . , 2001 ) , whereas a recent shotgun lipidomics study found about 10% pa of total lipids in whole cell extracts ( klose et al . these findings indicate that the short - lived intermediate pa may be metabolized during cell fractionation and may indeed be present in higher amounts than previously suggested . on the other hand , sole variation of the acyl - chain composition of pa in the liposome binding assay eliminated influences of membrane electrostatics and curvature as well as of proteins , such as the yeast vap scs2 , which is required for binding of opi1 to pa in vivo ( loewen et al . , 2004 ) . ( 1 ) currently , the mechanisms that regulate the total amount of cellular pls and their specific acyl - chain composition in response to altered fa supply are largely unclear . a mechanism , as described here , that responds not only to the content of critical lipid intermediates such as pa but also to its specific molecular composition provides an attractive regulatory circuit to integrate metabolic input parameters , such as availability of nutritional fas . ( 2 ) fa synthesis itself is under several levels of control and has a profound impact on metabolic and transcriptional regulation . ( 3 ) the major and highly conserved energy - sensing snf1 kinase operates through additional levels of regulation , namely , its metabolic downstream target acc1 . in conclusion , we demonstrate that acc1 hyperactivity leads to overproduction of fas and a shift in acyl - chain length distribution toward longer chains in all pl classes as well as in tag . the acyl - chain distribution of pa is critical for its interaction with the opi1 repressor , which displays reduced affinity for pa molecular species containing longer and more unsaturated acyl - chains . in the context of the snf1 mutant scenario , acc1 hyperactivity is a potent player in sequestering carbon into fat production and regulating cell growth and proliferation . this unveils an impact of the major energy - sensing kinase on membrane lipid composition and function and may explain some of the seemingly unrelated pleiotropic consequences of its deficiency . s. cerevisiae strains used in this study , except for the aid strain , are congenic with by , a derivative of s288c , and are listed in the supplemental experimental procedures . for liquid media experiments , cells were cultivated on a rotary shaker at 30c and 180 rpm using threonine - free minimal synthetic defined media with ( + i ) or without ( i ) 75 m inositol ( villa - garca et al . , 2011 ) . solid ypd ( 1% yeast extract , 2% peptone and 2% glucose , 2% agar ) and yeast extract peptone ( 1% yeast extract , 2% peptone , 2% agar ) with 2% of the nonfermentable carbon sources sucrose , glycerol , ethanol , or 1% glycerol and 1% ethanol were additionally used for plate tests . cell cultivation if not described differently was performed as follows ; a single colony of the desired strains was taken from a ypd plate and grown for 16 hr in + i medium ( 50 ml ) to midlogarithmic growth phase , rediluted to optical density 600 ( od600 ) = 0.1/ml in 100 ml of fresh + i medium , and grown for an additional 4.5 hr maintaining midlogarithmical growth . a cell aliquot of od600 = 20 was then shifted by filtration ( pore size , 0.45 m ) to 40 ml of fresh + i and i media , respectively , resulting in an initial od600 = 0.5/ml ( 0 hr time point ) . cell growth was monitored during growth , and doubling times were determined from the od600 readings as described elsewhere ( gaspar et al . , 2011 ) . for cultivation of the fa auxotrophic ole1 strain , the media for the 16 hr precultivation and the 4.5 hr growth after redilution contained 0.01% oleic acid and 1% tergitol . additionally , 20 ml of a 1 m stock solution of mes , ph 6.0 , was added per liter medium , resulting in an initial ph of 5.7 . cells were then shifted to fresh i media containing 20 mm mes with 0.01% oleic acid and 1% tergitol or with solely 1% tergitol , as a control , and grown for 6 hr . acc1 activity was inhibited in vivo by adding 1 g / ml sora to the culture medium where indicated : sora is highly specific for the eukaryotic form of acetyl - coa carboxylase , and by its binding to the biotin carboxylase dimer interface , it inhibits oligomerization , which is required for enzyme activity ( shen et al . , 2004 ) . for plate tests , a single colony of the desired strains was taken from a ypd plate and grown overnight in ypd medium ( for the ole1 strain , 0.01% oleic acid was supplemented ) to reach late - logarithmic growth phase . a cell aliquot of od600 = 10 was harvested and washed twice with sterile water . five microliters of serial 1:10 dilutions starting with od600 = 1/ml were spotted on to the indicated media plates . plates were analyzed after 2 days of growth at 30c . to determine overproduction and excretion of inositol ( opi phenotype ) , 5 l od600 ( 1 aliquot of wild - type , teto7acc1 , and the control strains opi1 and cho2 ) were spotted on i medium , containing the indicated concentration of doxycycline . cells were grown for 2 days at 30c and sprayed with a suspension of the inositol auxotrophic ( ino ) tester strain ( aid ) , which grows as a halo only around strains excreting inositol . for microscopic analysis , cultures were grown as described above and analyzed 3 hr after the shift or in stationary phase . labeling of neutral lipids with bodipy 493/503 or nile red was performed by adding the fluorescence dye directly to the cell culture for 10 min at room temperature in the dark ( final concentration , 1 g / ml ) . cells were grown as described above , and a cell aliquot of od600 = 20 was harvested at the indicated time points after the media shift . lipids were extracted according to a slightly modified folch method ( folch et al . , 1957 ; schneiter and daum , 2006 ) and analyzed on a uplc - synapt qtof hdms system ( waters ) , as described by knittelfelder et al . an aliquot of the lipid extract was used for methyl ester production and gas chromatography - mass spectrometry measurements . refer to supplemental experimental procedures for lipid extraction , chromatography , and mass spectrometry parameters . cultures were grown as described above and harvested 1.5 hr and 3 hr after the shift . real - time pcr was conducted as described elsewhere ( gaspar et al . , 2011 ) . ino1 values were normalized to wild - type grown on + i media for 1.5 hr following the shift ( ino1-repressing conditions ) . refer to the supplemental experimental procedures for rna isolation , reverse transcription , and real - time pcr parameters . for opi1 binding assays , 150 l of liposome suspension and 150 l glutathione s - transferase ( gst)-opi1 were incubated for 30 min at 22c and centrifuged at 30,000 rpm for 10 min at 22c in an optima tlx ultracentrifuge using a tla-100.3 fixed angle rotor ( beckmann ) . pellets were washed once with elution buffer without glutathione , resuspended in 150 l elution buffer without glutathione , and precipitated with 600 l acetone at 20c overnight . additionally , 150 l gst - opi1 were precipitated with 600 l acetone at 20c overnight as a loading control . precipitated gst - opi1 protein was pelleted at 13,200 rpm for 10 min at 4c in an eppendorf centrifuge ( type 5415r ) . pellets were washed once in 600 l acetone and dissolved in protein loading buffer for sds - page . for detection of gst - opi1 fusion protein , western blot analysis was performed using an anti - gst antibody from goat ( sigma - aldrich , 1:10.000 ) and an anti - goat antibody conjugated with horseradish peroxidase ( pierce , 1:7.500 ) for detection with supersignal west pico chemiluminescent substrate ( thermo scientific ) . additional experimental information in expanded form on acc1 in vitro mutagenesis , phospho - mass spectrometry , fluorescence microscopy , lipid analytics , ino1 expression , opi1 overexpression and purification , liposome studies , and pa 32:1 synthesis is available in the supplemental experimental procedures .

summarymembrane phospholipids typically contain fatty acids ( fas ) of 16 and 18 carbon atoms . this particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues . here , we show that the relative proportion of c16 versus c18 fas is regulated by the activity of acetyl - coa carboxylase ( acc1 ) , the first and rate - limiting enzyme of fa de novo synthesis . acc1 activity is attenuated by ampk / snf1-dependent phosphorylation , which is required to maintain an appropriate acyl - chain length distribution . moreover , we find that the transcriptional repressor opi1 preferentially binds to c16 over c18 phosphatidic acid ( pa ) species : thus , c16-chain containing pa sequesters opi1 more effectively to the er , enabling ampk / snf1 control of pa acyl - chain length to determine the degree of derepression of opi1 target genes . these findings reveal an unexpected regulatory link between the major energy - sensing kinase , membrane lipid composition , and transcription .

Introduction Results Discussion Experimental Procedures

the hydrophobic barrier of membranes is comprised of fatty acid ( fa)-derived acyl - chains , typically 16 or 18 carbon atoms in length ; these fas are synthesized in a cyclic series of reactions by the multifunctional fa synthase ( fas ) complex by the addition of c2 units that are derived from malonyl - coenzyme a ( coa ) ( smith et al . remarkably , the fa chain length in membrane phospholipids ( pls ) is evolutionarily highly conserved ( daum et al . apparently provides maximum stability to the otherwise fluid bilayer structure through their hydrophobic interactions . at the same time , this acyl - chain composition permits rapid and significant adjustments by a single elongation step ( c16c18 ) and/or desaturation ( c16:0c16:1 ; c18:0c18:1 ) in response to alterations in environmental conditions such as temperature . similar to mammalian cells , yeast membranes predominantly consist of mono- and di - unsaturated pls containing c16 and c18 acyl - chains to maintain the liquid crystalline state at physiological temperature ( de kroon et al . a decrease in the ambient temperature primarily triggers an increase in the c16/c18 ratio rather than a change in the degree of acyl - chain desaturation ( martin et al . the mechanisms that control the ratio between c16 versus c18 fas in cellular lipids are unknown . fas are synthesized de novo by the fas complex , which typically limits acyl - chain length extension to c16 and c18 carbon atoms , both in yeast and in mammals ( okuyama et al . , 1983 ) ; fas are subsequently channeled through the common precursor phosphatidic acid ( pa ) into the synthesis of neutral and polar lipid species , i.e. the substrate for the fas complex , malonyl - coa , is synthesized by the multifunctional enzyme , acetyl - coa carboxylase ( acc1 ) , the initial and rate - limiting step in cellular fa de novo synthesis in yeast ( al - feel et al . acc1 activity nutritional and metabolic signals , such as glucose limitation and salt stress , are transduced to acc1 by the snf1 kinase , the yeast ortholog of mammalian amp - activated protein kinase , ampk ( carling et al . , 1994 ; snf1 is the major energy - sensing kinase that phosphorylates and inactivates acc1 in vivo under conditions of low energy load ( woods et al . thus , yeast mutants lacking snf1 kinase display 3-fold elevated acc1 activity ; in addition , snf1 mutants are defective in transcription of multiple genes including ino1 , which encodes inositol-3-phosphate synthase and represents the most highly regulated gene of pl biosynthesis ( galdieri and vancura , 2012 ; jesch et al . notably , transcription of acc1 and pl biosynthetic genes , including ino1 , is strongly regulated by inositol in the growth media , which is mediated by the uasino element in the promoter of these genes ( carman and han , 2009 ; henry et al . full activation of ino1 transcription occurs in the absence of inositol and requires the ino2/ino4 transcriptional activator complex that binds to the uasino element ( ambroziak and henry , 1994 ; lopes and henry , 1991 ) . opi1 is a pa - binding protein and shuttles between the endoplasmic reticulum ( er ) and the nucleus , dependent on the level of pa and the presence of the yeast vamp - associated protein ( vap ) homolog scs2 , in the er . in the absence of inositol , pa levels are high , opi1 is bound to the er , and transcription of ino1 is enabled , leading to de novo production of inositol . in contrast , supplementation of inositol to the growth medium depletes pa by draining it into phosphatidylinositol ( pi ) synthesis , which promotes translocation of opi1 into the nucleus and repression of the ino1 gene ( loewen et al . defects in pl metabolism that lead to an increased steady - state concentration of pa in the er cause sequestration of the opi1 repressor to the er and elevated transcription of uasino - dependent genes . , 1982 ; we previously showed that the inositol requirement of the snf1 mutant is suppressed by downregulation of acc1 , suggesting that its activity might be connected to the ino2/ino4/opi1 regulatory circuit ( shirra et al . to uncouple acc1 activity from snf1-dependent phosphorylation , we have now characterized and mutated the specific phosphorylation site at position serine 1157 of acc1 . we provide evidence for a direct regulatory function of fa de novo synthesis in the transcriptional regulation of pl biosynthesis through the ino2/ino4/opi1 regulatory circuit . we show that acc1 activity is a critical determinant of the c16/c18 acyl - chain ratio in cellular lipids ; in conjunction with the specific binding preference of the opi1 repressor for c16-pa species , this describes a regulatory mechanism of gene transcription in response to altered acyl - chain length in cellular lipids and unveils a function of the snf1/amp - activated protein kinase in regulating lipid homeostasis . as shown in figure 2a , the acc1 mutant , generated in this fashion , grew like wild - type on complete yeast extract peptone dextrose ( ypd ) media , but similarly to the snf1 mutant , it was resistant to the acc1-specific inhibitor , soraphen a ( sora ) ( shen et al . this inositol auxotrophy ( ino phenotype ) could be suppressed either by decreasing acc1 activity with sora ( figure 2a ) or in the snf1 mutant by downregulation of acc1 transcription under control of the heterologous doxycycline - repressible tet07 promoter ( teto7-acc1 ; figure s2a ) . notably , the acc1 mutant was capable of growing on sucrose as the sole carbon source or on nonfermentable carbon sources like glycerol or ethanol , which distinguishes its phenotype from that of snf1 mutants with respect to carbon source utilization ( figures s2b and s2c ) . most notably , as a result of elevated malonyl - coa production by hyperactive acc1 in the acc1 mutant , fa acyl - chain distribution was significantly shifted toward longer acyl - chain lengths , increasing the c18:c16 acyl - chain ratio from 0.5 in wild - type to 2.0 in the acc1 mutant ( figure 2d ) . this shift is also reflected in the altered acyl - chain composition of virtually all glycerolipid classes for instance , tag ( figure s2e ) . taken together , the acc1 mutant displays similar phenotypes as the snf1 mutant with respect to fa overproduction , a shift to longer acyl - chains , and tag accumulation , as well as inositol auxotrophy . wild - type cells treated with the same concentration of sora , on the other hand , exhibited a severe growth defect in both the presence and absence of inositol , which is consistent with the essential role of acc1 in cellular metabolism ( figure 3a ) . from previous studies , it is known that ino1 expression responds to the localization of opi1 , the transcription factor that senses the levels of the central lipid intermediate , pa , in the er ( loewen et al . notably , inhibition of acc1 activity in the acc1 mutant readily promoted export of opi1-gfp from the nucleus , leading to the observed relief of ino1 expression ( figures 3b and 3d ) . since opi1 localization responds to the amount of pa in the er ( loewen et al . , 2004 ) , we hypothesized that overproduction of fa might lead to reduced cellular pa levels , perhaps due to its increased channeling into tag synthesis . , that reduced pa levels are responsible for translocation of opi1 from the er into the nucleus to repress ino2/ino4-dependent ino1 transcription ( loewen et al . since the molecular species distribution in all glycerolipids displayed a characteristic shift toward increased acyl - chain length in acc1 , we hypothesized that not the total amount of pa but rather its specific molecular acyl - chain composition may define its binding affinity to opi1 . thus , we next analyzed the molecular species composition of pa ( and of the major cellular pl phosphatidylcholine [ pc ] as a reference ) in wild - type and acc1 strains in greater detail . as shown in figure 5 , pa 32:1 ( containing c16:0 and c16:1 acyl - chains ) and pa 32:2 ( containing two c16:1 acyl - chains ) species were indeed greatly reduced in the acc1 mutant , whereas pa 36:1 ( containing c18:0 and c18:1 acyl - chains ) and pa 36:2 ( containing two c18:1 acyl - chains ) species were increased . this shift in acyl - chain composition was reversed by the addition of sora , a condition that also led to inositol prototrophy ( figures 2a and 3a ) , derepression of ino1 ( figure 3b ) , and nuclear export of opi1-gfp ( figure 3d ) in the acc1 strain . based on the hypothesis that an increase in the proportion of c18 versus c16 acyl - chains in pa is responsible for nuclear localization of opi1 , we next tested whether exogenous c18:1 supply and concomitant changes in the acyl - chain composition would phenocopy the effects of the hyperactive acc1 allele also in wild - type cells . similarly , the ole1 mutant , which lacks the sole fa desaturase in yeast ( stukey et al . , 1989 ) , and which is dependent on unsaturated fa supplementation , displayed a strict inositol auxotrophy in the presence of c18:1 . this rescue of the acc1 mutant growth was not due to repression of acc1 activity by c16:1 , since total c18 fas remained at a high level , characteristic for acc1 hyperactivity ; rather , the ratio between c16 and c18 fas was restored by c16:1 supplementation ( figure 6b ) . the analysis of pa species in wild - type and ole1 strains supplemented with c18:1 showed a similar shift toward longer acyl - chain lengths as in the acc1 strain as compared to wild - type grown without c18:1 ( figure 6c ) : pa 32:1 and pa 32:2 species were significantly decreased , whereas pa species with longer fa chain length increased , especially 36:2 . to further confirm whether the acyl - chain composition of pa affects the interaction with opi1 , we performed in vitro binding assays with pc 36:2 liposomes as a matrix , containing 20 mol% of different pa species . indeed and in line with our hypothesis , we found significantly different binding affinities of opi1 for pa species dependent on the acyl - chain composition . liposomes containing saturated pa species ( pa 32:0 and pa 36:0 ) showed the highest affinity , followed by liposomes containing 32:1 pa ; liposomes containing pa 36:2 showed the lowest binding affinity for purified opi1 . these results clearly demonstrate that the acyl - chain composition of pa is a major determinant of its interaction with the transcriptional repressor opi1 . this preferred binding affinity explains the observed phenotypes and significantly extends the current model of the transcriptional regulation of pl biosynthesis in yeast in response to deregulated fa de novo synthesis . throughout the eukaryotic kingdom , fas of 16 or 18 carbon atoms in length are the preferred acyl components in all glycerolipids that serve as bulk lipids in biological membranes or as storage lipids . whereas mechanisms controlling the level of fa desaturation in response to ambient temperature and membrane fluidity have been described in detail , much less is known about the mechanisms regulating chain length distribution ( de kroon et al . cellular fa synthesis is also coordinated with nutritional supply , activation , and incorporation into complex lipids to support proper cell function and to prevent potentially detrimental fa - induced lipotoxic effects , which are believed to be causative for lipid - associated disorders , such as obesity and type 2 diabetes in humans ( kim and ye , 2013 ; unger , 2002 ; vidal - puig and unger , 2010 ) . thus , endogenous de novo synthesis of particular fas also plays an important role to counterbalance the physiological impact of nutritional fas that may be incorporated into cellular lipids . in yeast and mammalian cells , acyl - chain length is largely determined by the fas complex that limits acyl - chain length to 16 and 18 carbon atoms ( okuyama et al . we now show that the activity of acc1 , the initial and rate - limiting enzyme of de novo fa synthesis , is a major determinant of total cellular fa production and also has a fundamental impact on the relative distribution of c16 and c18 acyl - chains in cellular lipids . on the other hand , the shift in acyl - chains toward longer residues is reflected in all cellular glycerolipids , with consequences for the binding and sequestration of the transcriptional repressor opi1 to the er . one level involves the expression of the acc1 gene , which is coordinately regulated with pl synthesis by the pl precursors , inositol and choline , and the positive and negative transcription factors ino2/ino4 and opi1 , respectively ( hasslacher et al . in addition , the activity of acc1 is strongly regulated by snf1-dependent phosphorylation ( mitchelhill et al . , 1994 ) , which , in turn , depends on the energy status of the cell ( wilson et al . here , we made use of a yeast mutant that lacks the single snf1 phosphorylation site in acc1 at serine 1157 ( acc1 ) and exhibits phenotypes very similar to snf1 mutants with respect to tag and lipid droplet accumulation as well as inositol auxotrophy , demonstrating that these snf1 phenotypes are caused by acc1 hyperactivity . the evidence we present here strongly supports the hypothesis that the inositol - requiring phenotype of the acc1 strain is due to decreased ino1 expression that is controlled by the well - characterized ino2/ino4/opi1regulatory circuit ( henry et al . , 2004 ) , as both deletion of opi1 , which uncouples transcriptional repression of ino1 from cellular pa levels , and overexpression of ino1 fully restored inositol prototrophy to the acc1 mutant strain . most notably , neither increased endogenous fa production nor exogenous fa supply led to increased pl accumulation , despite the fact that an altered acyl - chain composition is reflected in all pl classes ; instead , excess fas are preferentially channeled into tag . in addition , the similarity of the cellular response to these two situations suggests that functional tag synthesis is crucial for accommodating these fas , thus avoiding the buildup of potential signal molecules , such as pa or dag , or free fas ( garbarino and sturley , 2009 ; kohlwein , 2010 ; kohlwein and petschnigg , 2007 ; petschnigg et al . , importantly , our evidence significantly extends the current model that indeed the acyl - chain composition of pa is the critical determinant of opi1 sequestration to the er . we show that alterations in the acyl - chain composition of the acc1 strain or of wild - type and the ole1 mutant in the presence of oleic acid lead to inositol auxotrophy due to the reduced binding affinity of the opi1 repressor to pa molecular species containing longer acyl residues . we hypothesize that the recognition of the negatively charged head group of pa by the basic tract of opi1 might only represent the first level of interaction , whereas the second level is presumably determined by sensing the nature of the acyl - chains of pa within the er membrane . several other studies also support the importance of acyl - chain composition of pa for interaction with proteins ( kooijman and burger , 2009 ) ; for example , the binding specificity of human protein phosphatase 1 ( pp1c ) to pa species harboring a higher degree of acyl - chain unsaturation ( jones and hannun , 2002 ) . these findings indicate that the short - lived intermediate pa may be metabolized during cell fractionation and may indeed be present in higher amounts than previously suggested . on the other hand , sole variation of the acyl - chain composition of pa in the liposome binding assay eliminated influences of membrane electrostatics and curvature as well as of proteins , such as the yeast vap scs2 , which is required for binding of opi1 to pa in vivo ( loewen et al . ( 1 ) currently , the mechanisms that regulate the total amount of cellular pls and their specific acyl - chain composition in response to altered fa supply are largely unclear . a mechanism , as described here , that responds not only to the content of critical lipid intermediates such as pa but also to its specific molecular composition provides an attractive regulatory circuit to integrate metabolic input parameters , such as availability of nutritional fas . ( 3 ) the major and highly conserved energy - sensing snf1 kinase operates through additional levels of regulation , namely , its metabolic downstream target acc1 . in conclusion , we demonstrate that acc1 hyperactivity leads to overproduction of fas and a shift in acyl - chain length distribution toward longer chains in all pl classes as well as in tag . the acyl - chain distribution of pa is critical for its interaction with the opi1 repressor , which displays reduced affinity for pa molecular species containing longer and more unsaturated acyl - chains . this unveils an impact of the major energy - sensing kinase on membrane lipid composition and function and may explain some of the seemingly unrelated pleiotropic consequences of its deficiency . acc1 activity was inhibited in vivo by adding 1 g / ml sora to the culture medium where indicated : sora is highly specific for the eukaryotic form of acetyl - coa carboxylase , and by its binding to the biotin carboxylase dimer interface , it inhibits oligomerization , which is required for enzyme activity ( shen et al . to determine overproduction and excretion of inositol ( opi phenotype ) , 5 l od600 ( 1 aliquot of wild - type , teto7acc1 , and the control strains opi1 and cho2 ) were spotted on i medium , containing the indicated concentration of doxycycline . refer to the supplemental experimental procedures for rna isolation , reverse transcription , and real - time pcr parameters .

the online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users . life expectancy of man , and especially man in the western world , increased by more than 2 days per week for the whole previous century . much of this dramatic increase is to the credit of hygiene , but medicines , and especially antibiotics and vaccines , have contributed significantly too . in the first world war , for example , almost as many soldiers died of disease as of bullets . during the second world war this unfortunate situation got medical doctors around the world can write prescriptions for tens of thousands of medicines , and an even larger number is available of herbal medicines , homeopathic wonder - cures , and other preparations for which the medicinal value has not been proven . most medicines function by interacting with proteins in the body . of the more than twenty thousand protein types in our body this , of course , gives hope for the future of drug design because most proteins are still available as a target for which a blockbuster drug can be designed . despite massive , world - wide efforts the number of new molecular entities ( nmes ) that the fda approves per year for use as medicines certainly is nt growing , while the amount of money involved goes up much faster than inflation even when we include obama s troubled asset relief program this journal ( jcamd ) has published many , many articles on techniques that according to the authors of these articles were the holy grail for drug design , and that in today s reality are just good tools used in this process . following a path familiar in science others follow suit and publish improvement after improvement , after which yet others start testing all similar methods . this was introduced in 2000 and only 3 years of improvements were needed before the first comparison methods were published . . the ever increasing costs mainly result from development and marketing and , unfortunately for us , not from research . this might explain why each time a new drug design research tool gets published pharmaceutical industries immediately jump on it and give it a hype status.fig . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone amount of money spent in billion us$/nme ( after munos ) . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone the first hype in drug design was born out of the famous article by hol in which he coined the name rational drug design for all protein - structure based techniques , thereby implicitly calling all methods that actually worked , such as screening or luck , irrational ; see fig . next to a monochrome picture showing this same fit , hol wrote : as to whether a drug can actually reach its target , e.g. the active center of an enzyme , is primarily a spatial problem . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . next to a monochrome picture showing this same fit , hol wrote : as to whether a drug can actually reach its target , e.g. the active center of an enzyme , is primarily a spatial problem . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . it is not by eye that we can determine either the fitness of a ligand for a pocket , or the safety or efficacy of a drug . it does not seem illogical to assume that the founders of jcamd were at least subconsciously dealing with the oversimplification implied by fig . we believe that most articles published in jcamd have dealt with aspects of drug design left out of fig . 2 . the advent of faster computers like first the vax / vms , then supercomputers such as the cray , and finally the pc , have allowed scientist to numerically solve chemical problems of ever increasing size and complexity . semi - empirical quantum calculation methods have been devised to calculate the chemically relevant aspects of the electronic wave - functions associated with small organic molecules and thus compute their 3d dimensional structures as well as the energy of their conformers [ 1519 ] . all the techniques derived in this domain are referred to as ligand - based drug design . in parallel , the development of molecular mechanics force fields combined with the fact that newton s equations of motion could be solved for entire proteins in their aqueous environments were true innovations in the investigation of the structure function relationships [ 2028 ] . thus , not only the geometry and the potential energy surface of macromolecular assemblies could be calculated but also their dynamic and thermodynamic properties [ 29 , 30 ] . for the early computational chemists this opened the perspective of testing at will the energy of interactions between protein targets and large collections of small molecule ligands [ 29 , 31 , 32 ] . the original thoughts that this would replace experimental validation processes , though , have long been shown to be a nice dream at best . the perception that the underlying mechanism of protein ligand recognition would be unravelled and would thus allow what ever since has been called structure - based drug design has never looked so clear and promising as at that particular moment in 1986 . with the exception of a very small fraction of ligands that are purely rigid the values of the torsion angles in ligands are determined by the valence electrons of the atoms . the development of empirical molecular mechanics force field in the late 1970s [ 33 , 34 ] have allowed for the in silico determination of the geometries ( low energy conformers ) of ligands in vacuo . application of these methods relies on two underlying assumptions : ( 1 ) that the conformation of the dissolved ligand corresponds closely to its gas - phase conformation ; and ( 2 ) that the biologically active conformation of the ligand is likely to be found among the set of low energy conformers of the isolated ligand [ 36 , 37 ] . the combined knowledge of the ligand structure ( determined by nmr or x - ray ) , the measured binding affinities , and the spatial overlay of the low energy conformations should then be sufficient to establish a structure activity relationship and pinpoint the spatial organization of the recurrent chemical features correlated with activity ( pharmacophore ) . this paved the way for a series of successes for ligand - based drug design [ e.g. 39 , 40 , 16 ] . however , although it seems fairly reasonable at first sight , both assumptions in practice proved to be incomplete and/or insufficient [ 4150 ] . the computational process by which the complementary aspects between a ligand and a receptor binding site can be ascertained has been explored with the design of specifically dedicated docking programmes . early docking methods were based uniquely on assessing the shape complementarity between a pocket in the 3d structure of a protein and low energy conformers of a ligand . the approach was computationally cumbersome due to the need to systematically search all possible ligand orientations within the pocket and scoring each of these poses by its steric hindrance . subsequent developments have taken place in several directions : improved scoring functions [ 5263 ] different ways to deal with ligand flexibility [ 60 , 6472 ] , and most recently also ways to deal with receptor flexibility [ 7378 ] . fundamental research has been performed into directions such as desolvation energies [ 7983 ] , or other aspects of the force fields used for scoring docking poses [ 66 , 78 , 8496 ] . the idea to calculate from first principles all atomic motions occurring in an active enzyme in its aqueous environment has attracted many scientists to computer aided molecular design . starting with the atomic loci obtained from the x - ray structure of en enzyme a series of snapshots describing the trajectory of the enzyme over time could thus be produced and ensemble average properties calculated based on boltzmann s ergodic hypothesis . the near infinite computer time needed for such experiments muted this field till concepts from alchemy could be embraced . in silico , one is not bound by the sequential order of events that govern paths between states , and hence so - called thermodynamic cycle methods could be developed that replaced chemical steps with alchemical steps that in principle should lead to the same outcome [ 29 , 30 , 97 , 98 ] . comparative molecular field analysis ( comfa ) is based on the overlay of active ligands [ 99 , 100102 ] . initially , the technique was more a concept than an effective tool as computer power was very limited and molecular descriptors as well as dedicated algorithms needed to be developed . the underlying idea that the 3d dimensional steric / non - bonded ( van der waals ) and electrostatic potential fields generated by the spatial organization of the chemical features around the scaffold of a ligand ( fig . 3 ) play a fundamental role in the biomolecular receptor recognition was so intuitively right and the technique made a break - through in 1988 . about 15% of all articles in jcamd refer to the use of this technique , refined and applied in all sorts of ways to produce the overly famous quantitative structure activity relationship ( qsar ) equations . however , comfa suffers from three drawbacks : ( 1 ) the alignment of the ligands in the pocket must be either known or gambled correctly ; ( 2 ) the method has been established for rigid or quasi - rigid classes of molecules ( e.g. steroids ) ; and ( 3 ) the detailed influence of the protein pocket is not known which means that any feature that is not implicitly present in the training set will be missed [ 104109 ] . these nearly fatal drawbacks prevented the generalization of the method as a standalone solution to rational drug design . certainly , the best way to apply comfa is to combine it with a pharmacophore model and a carefully conducted conformational study of the ligands .fig . 3contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups many drug design projects include at some stage knowledge of the 3d structure of the target protein , and homology modelling is normally used when neither x - ray nor nmr derived coordinates are available [ 111118 ] . many computer programs were written for this purpose [ 119123 ] and the casp competition illustrates every 2 years where the field stands . presently , yasara seems to be performing very well , but many labs are working hard so this situation might change again in the future . for example , methods are under development that use plim to provide a first fix on the ligand docking site where - after steered molecular dynamics is used to continue the trajectory to convergence . similar to the casp competitions , the gpcr - dock [ 126 , 127 ] competitions have evaluated the quality of docking software , but with the additional complexity that the target structures needed to be modelled before docking could be attempted . in recent years a whole series of studies have been published in which homology modelling , combined with other tools , proved a viable replacement for the cumbersome experimental determination of target structures [ 111 , 114 , 128 , 129 ] . the good performance of two dutch teams in the recent gpcr dock competition beautifully illustrates the often mentioned fact that even the best tools only perform well in the hands of good scientists . in this latter article we find the interesting quote interestingly enough , it is the model built with most human intervention which proves to be the best . in the early 1990s the radical new idea emerged that instead of the virtual and/or real screening of large libraries of already existing molecules to identify new bioactive hits , one could rather attempt to construct entirely new synthesizable molecular entities solely based on the knowledge of the active site of the pharmaceutical target enzyme . to do so , small organic fragments composed of few atoms only must be assembled in silico inside the binding sites of enzymes in such a way that optimal protein ligand , steric , and electronic complementarity is achieved [ 84 , 125 , 135141 ] . the major problem of this approach arises from the complexity of the active site landscape and the combinatorial vastness of all possible arrangements of fragments in the volume delineated by an enzyme active site [ 66 , 142144 ] . how to choose the first fragment and where should it be positioned and oriented with respect to the inner surface of the binding pocket or cleft [ 143 , 145 , 146 ] ? which next fragment should be attached to it ? ? the genetic algorithms [ 66 , 142 , 148150 ] have been invented which allow this concept to be realized within a tractable amount of computer time by performing random transformations on a ligand collection . these transformations are selection , mutation , and crossover , and are reminiscent of the corresponding evolutionary processes in biology underlying the optimisation of genes , hence their name genetic algorithms. experience shows that these algorithms provide solutions that nicely fit the objective function , although it often is difficult to understand exactly why . randomly screening very large libraries containing up to 10 or even 10 chemical entities in in vitro enzymatic assays to produce leads has been the central paradigm of the pharmaceutical industry across the 1990s . however , after years of operating very expensive screening facilities , it has been realized that the hits produced were not of the expected quality . for example , often a bias is observed toward too lipophilic compounds that are impossible to optimize . compared to the actual number of chemically entities ( ~infinite ) the any amount of compounds that can be screened via this process is essentially zero [ 151 , 152 ] . in parallel , computational chemists had inferred that screening could be successfully operated virtually throughout computers at all stages in the drug design process from hit identification via hit optimization to lead optimization [ 153162 ] . in each of these three stages virtual libraries can be created and filtered either using chemometrics to exclude molecules that obviously are nt drug - like because of their predicted solubility or adme / tox properties , or using 3d chemical molecular descriptors ( pharmacophores ) , or using docking results . thus , libraries of compound that do not actually exist can be screened and a much smaller , manageable number of compounds selected . this is of particular advantage at the stage of lead optimization , when only few compounds are left . scaffolds of lead compounds usually carry a number of branching points were chemical variation is allowed . the in silico creation of combinatorial libraries of all the variant compounds is a dramatically faster process than its in vitro counterpart [ 163165 ] . one of the main difficulties in establishing reliable and/or transferrable qsar equations is that , even within a class of chemical analogues , ligand affinities may not respond linearly to the variation of one or several of the molecular descriptors that have been identified as related to activity . for instance , across a series of chemical substituents sorted by increasing polarity the measured affinity may respond linearly only for a restricted number of them because steric hindrance or global effect such as desolvation may penalize the binding of slightly larger groups . the modification of a branched group at another point around the scaffold may however allow some of the previously excluded ligands to become highly active . indeed the mere addition of one methyl group may result in a sudden tenfold leap in potency , dramatically increasing ligand efficiency [ 166 , 167 ] . it was demonstrated that these problems could be circumvented using artificial intelligence methods ( neural network , support vector machine , etc . ) that are insensitive to the spatial alignment of the ligand scaffolds and that are able to recognize particular combinations of properties distributed around the scaffold of a set of active ligands [ 168170 ] . artificial intelligence can be ubiquitously implemented at various stages in the rational drug design process to improve results that can be otherwise be more uncertainly obtained with classical methods , especially when assessing general properties that are the result of the subtle combination of many different factors in relation to others such as drug - likeness . various examples of artificial intelligence applications and their limitations have been published in jcamd [ 172175 ] . notwithstanding the utility of artificial intelligence , normal intelligence remains useful in avoiding some of the all too common pitfalls in the derivation and application of qsar models . we apologise to the many authors of methods that did nt make it into the above list ( see esm table 1 ) . much good work has been done that the editors certainly would nt allow us to include because citing all 1,200 articles published in jcamd in the first 25 years would perhaps be a bit excessive . we could have mentioned the work by che on privilege structures , or by lotta et al . on multivariate pls modelling of data sets . the recent work by zhou et al . on the use of dft calculation to accurately assess the existence of intermolecular h - bonds in docking instances . sarmah et al s work on solvent effects also added significantly to the drug - designers toolbox , but the methods described in these articles did nt achieve hype status . the rapid increase in costs of developing and marketing new medicines is not leaving the pharmaceutical industry untouched . recent years have seen a strong concentration of activities in terms of mergers , buy - outs , and closures . it may simply be , that a research - intensive industry like the pharmaceutical industry does not lend itself to the type of management that is common in consumer goods , fashion and footwear . it seems a paradox , though , that the high costs associated with drug design are caused by development , marketing , and legal fees , but when it comes to cost - reduction research departments are , euphemistically called , consolidated . jcamd has published a large series of articles in which multiple methods have been combined . [ 22 , 128 , 129 , 181185 ] . all these pipelines and otherwise combined methods speed up the use of the existing tools , and allow them to be applied to ever larger numbers of small molecules in ever shorter times . actually , there is a new hype raging at the moment , and it is called translational science. in the wikipedia we find under translational research : in the field of medicine , for example , it is used to translate the findings in basic research more quickly and efficiently into medical practice and , thus , meaningful health outcomes , whether those are physical , mental , or social outcomes . in a sense , the recent spate of articles on combining existing techniques into more easily applicable super - tools fit nicely to this translational paradigm . it must be stated , though , that the translation science hype is feeling stiff competition from systems biology and modelling pharmacokinetics and pharmacodynamics . between the lines we read in translational science that the pharmaceutical industry has finally realized that our deep lack of understanding of all aspects of the interaction of a medicine with a human being is the main cause for luck still being the most determining factor in the drug design process . consequently , we see the out - sourcing budgets of the large pharmaceutical industries go up , and more and more fundamental research performed in academia is finding its way to small and medium size enterprises ( smes ) where it can be incorporated in their lean and mean research machines . big pharma will at some time buy either their products or the whole smes and convert validated targets and leads or even phase i products into new medicines . this new paradigm will probably also be proven a hype soon ; only time can tell if translational research will rescue the pharmaceutical industry , or that it will only better illustrate what it all is that we do nt know yet . it remains a fact that better understanding the underlying biology , better treatment of all available data , and more intelligent combinations of data , information , and knowledge must be beneficial for the drug design process and thus , on the long run , for all of us . if the pharmaceutical industry wants academia more involved in the drug design process they could themselves make a giant first step by making available all ( or at least very many ) x - ray structures of protein ligand complexes . we estimate that the number of pdb [ 189 , 190 ] files collecting computer dust in the pharmaceutical industry is considerably bigger than the 75,000 structures now in the pdb . we have discussed this possibility with industrial crystallographers who realized that they were sometimes sitting on thousands of structure files for which secrecy was no longer an issue . they remained nevertheless hesitant to even consider discussing with their management the release of these data in fear of paranoia based rejection . another often heard rejection criterion is that they are a bit ashamed for these data because often these files have not been refined any further than was needed to answer the biological or pharmaceutical question at hand . we offer to set up a database for these files , and we offer to re - refine all industrial structures of protein ligand complexes . we will then only release those coordinates to the wider public that pass certain minimal validation criteria . if one day deposition of coordinate files into the pdb becomes significantly easier , we can consider depositing all files in the pdb on behalf of the original depositors . it might seem a bold promise to re - refine perhaps even 100,000 structures , but the pdb_redo experiment [ 192196 ] shows that today this can be done . in pdb_redo we significantly improved 85% of all presently available pdb files that were solved by x - ray . it seems likely that structures that often have been minimally refined can be improved even easier . one can even envisage that industries would like to look back at their own coordinates after we went through the elaborate and time consuming refinement process for them ; in management speak that would be the ultimate win win situation . despite massive efforts in the design of tools , databases , robotic techniques , and management innovations , luck seems to be at the basis of the discovery of most new medicines . the blockbuster viagra is probably the best illustration of the opportunism that we tend to call serendipity . in 1997 , i.e. , long before the first gpcr structure became available , kuipers et al . performed a massive literature search for aryloxypropanolamines and similar compounds binding to the serotonin 5ht-1a receptor and a series of sequence similar amine receptors . a correlation analysis revealed that only one residue s presence / absence showed a perfect correlation with binding / non - binding of a series of compounds . a mutational study validated the hypothesis that this correlation indicated a direct hydrogen bond between an alcohol group in the aminergic ligand and asparagine 719 . when the structure of the human 2 adrenoceptor bound to carazolol was solved by x - ray [ pdbid 2rh1 ; 202 ] , it showed indeed two hydrogen bonds between asn-719 and this similar ligand ( see fig 4 ) . by the way , in none of the gpcr homology models available in 199 , left : ( part of ) the x - ray structure of the 2 adrenoceptor bound to an inverse agonist that is somewhat similar to ( s)-penbutolol . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available ligand binding by asn-386 . left : ( part of ) the x - ray structure of the 2 adrenoceptor bound to an inverse agonist that is somewhat similar to ( s)-penbutolol . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available in another gpcr related project aimed at using as much heterogeneous data as can possibly be combined , oliveira et al . predicted the role of all active site residues in gpcrs , the pivotal role of arg-340 , and even a series of residue interactions involved in the activation process , and the presence and location of helix viii . the recent flurry of articles on gpcr xray structures [ 206209 ] , and especially the structure with a covalently agonist - bound g protein showed all these predictions to be conceptually right . these two gpcr - related examples make clear that there is a lot to be gained from using experimental data . but these examples also taught us how hard it is to actually get access to those data . with the gpcrdb [ 211213 ] we have started a trend to make molecular class specific information systems ( mcsis ) . and a small company , bio - prodict ( www.bio-prodict.nl ) recently caught on and is now making mcsises for a wide variety of commercially interesting molecules [ 214218 ] . their systems ( some of which are freely accessible from their website ) revolve around a structure based , and thus very accurate multiple sequence alignment ( msa ) for a whole protein super - family . this msa then functions as the anchor on which to position all kinds of data that can range from 3d structures to genome related data , from mutation studies to ligand binding constants , or from sequence correlation patterns to the prediction of mutations that enhance the protein s stability . as the most powerful information tends to be carefully hidden in the literature , an extensive set of literature - mining scripts aids with the extraction of , for example , mutation information . in fact , it was shown that the suite of mutation data extracting scripts reaches a much better coverage than can be obtained by human experts [ 214218 ] . a recent development that will aid the drug hunters of the future . showed how this programmable pdf reader could be used to directly couple data in articles on gpcrs to the gpcrdb . first , the residue numbering problem gets solved because the reader can ask the gpcrdb for the position in the gpcr msa of any residue mentioned in the article , and it can even modify or correct the sequence numbers in the article if needed . much good gpcr mutation data was published in the pre - gpcr - structure era that ended with the opsin structure article , and often these data were misinterpreted because of the poor quality of the available homology models . the utopia - gpcr pdf reader can correct those interpretations thereby salvaging old , high quality experimental data for future use . figure 5 shows an image from an old mutation study in which the authors describe several ground - breaking mutations in the guinea pig histamine h1 receptor , building and validating a homology model using these data , and arguing , for example , that residue trp161 plays an important role in receptor - ligand binding . this assumption was based on the effect of the mutation on receptor function , leading to a model in which trp161 was modelled in the ligand - binding site . by contrast , the gpcrdb generated annotation listed in the sidebar of the reader indicates that this residue , located in tm iv , points towards the membrane and possibly interacts with cholesterol . looking at the model provided by the gpcrdb , based on the latest crystal structures , it can be seen that a direct role of trp161 in receptor - ligand binding is highly unlikely.fig . 5left , one page from the histamine h1 article by wieland et al . in which trp161 is suggested to interact with the ligand while the pdf reader sidebar shows today s interpretation that this tryptophan is facing outwards towards the lipid or a dimer partner . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . the gpcrdb does not ( yet ) dock ligands , so the ligand is represented by a hand - added gray ball left , one page from the histamine h1 article by wieland et al . in which trp161 is suggested to interact with the ligand while the pdf reader sidebar shows today s interpretation that this tryptophan is facing outwards towards the lipid or a dimer partner . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . the gpcrdb does not ( yet ) dock ligands , so the ligand is represented by a hand - added gray ball folkerstma et al . combined with manually curated multiple sequence alignments , key positions in the ligand binding pocket were identified that had specific interactions with functionally diverse compounds . this analysis required a substantial amount of work : categorizing structures and compounds , creating multiple sequence alignments , analyzing ligand contacts , and transferring the results into a homogeneous residue numbering scheme ( the so - called 3d numbers ) . with the 3 dm information system [ 223 ; see the help movie ] , these analyses can today be performed in a matter of minutes [ 215 , 217 , 224].fig . 6bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds more than 100 articles were found that discuss the effects of mutating this residue on the ligand binding of the receptor . in all these articles the use of a common 3d numbering scheme enables transfer of heterogeneous information between protein family members . figure 7 shows 40 antagonists in red and 70 agonists in blue . in this example , a hundred articles had to be read to extract all available mutation information for this single position mutated in 22 different receptor species combinations . that these 100 articles had to be found among 100,000 pubmed entries that contain nr information 7cartoon representation of two superposed representative nr structures ( one bound to an agonist ; one bound to an antagonist ) . the ligands were placed in the same orientation as found in their native pdb file . residue 26 , for which the antagonist interaction had been mentioned in the literature , is shown in yellow , as is residue for which fig . what if suite cartoon representation of two superposed representative nr structures ( one bound to an agonist ; one bound to an antagonist ) . the ligands were placed in the same orientation as found in their native pdb file . residue 26 , for which the antagonist interaction had been mentioned in the literature , is shown in yellow , as is residue for which fig . what if suite if , one day , all structures of nr - ligand complexes that now are scattered over inaccessible industrial hard disks could be concentrated in one system , then we could consider asking much more elaborate questions . we could consider correlating aspects of ligands with protein atom characteristics , or we could analyse if residues not contacting the ligand have an influence on binding or activation , etcetera . it is not only important to get as much information as possible stored in systems amenable to scrutiny , but it is also important to realize that for every one bioinformatician or drug hunter there are one hundred scientists who do not use molecular software regularly . project hope aims to predict the molecular phenotype of point mutations that were shown causally related to human disease states . this system attempts in all stages of user interaction to cater for human geneticists who typically do not use molecular software at all . hope only asks the user to cut - n - paste the sequence , and click the residue mutated and the mutation residue type . it then builds a homology model if needed , calls dozens of servers and services in seven countries , combines all possible information and writes a final report that can be directly used in publications , but , more importantly , that is written without using any bioinformatics jargon and even has a build - in dictionary that explains terms such as active site , salt - bridge , or hope thus is the ultimate translation machine because in doing translational research it even translates between the researchers . we believe that the recent spate of consolidations in the pharmaceutical industry is not a problem but an opportunity . mankind needs medicines , and now that pushing ones luck is slowly becoming a less successful technique , only research can find them . this research can progress rapidly if the thousands and thousands of x - ray structures of protein ligand complexes would find their way from hard - disks behind pharmaceutical industry firewalls to the public domain . drug design research in the next 25 years will revolve around ever broader collaborations , ever more holistic understanding of the drug human interactions , and ever better use of the available data , information , and knowledge . supplementary material 1 ( docx 89 kb ) supplementary material 1 ( docx 89 kb )

in its first 25 years jcamd has been disseminating a large number of techniques aimed at finding better medicines faster . these include genetic algorithms , comfa , qsar , structure based techniques , homology modelling , high throughput screening , combichem , and dozens more that were a hype in their time and that now are just a useful addition to the drug - designers toolbox . despite massive efforts throughout academic and industrial drug design research departments , the number of fda - approved new molecular entities per year stagnates , and the pharmaceutical industry is reorganising accordingly . the recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside . the next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved , from quantum chemistry to bedside and from academia to industry . this will above all include understanding the underlying biological problem and optimal use of all available data.electronic supplementary materialthe online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction A brief history of tools Where do we stand today? Dealing with data, information, and knowledge: from hype to hope? Electronic supplementary material

the online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users . life expectancy of man , and especially man in the western world , increased by more than 2 days per week for the whole previous century . much of this dramatic increase is to the credit of hygiene , but medicines , and especially antibiotics and vaccines , have contributed significantly too . during the second world war this unfortunate situation got medical doctors around the world can write prescriptions for tens of thousands of medicines , and an even larger number is available of herbal medicines , homeopathic wonder - cures , and other preparations for which the medicinal value has not been proven . of the more than twenty thousand protein types in our body this , of course , gives hope for the future of drug design because most proteins are still available as a target for which a blockbuster drug can be designed . despite massive , world - wide efforts the number of new molecular entities ( nmes ) that the fda approves per year for use as medicines certainly is nt growing , while the amount of money involved goes up much faster than inflation even when we include obama s troubled asset relief program this journal ( jcamd ) has published many , many articles on techniques that according to the authors of these articles were the holy grail for drug design , and that in today s reality are just good tools used in this process . this might explain why each time a new drug design research tool gets published pharmaceutical industries immediately jump on it and give it a hype status.fig . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone the first hype in drug design was born out of the famous article by hol in which he coined the name rational drug design for all protein - structure based techniques , thereby implicitly calling all methods that actually worked , such as screening or luck , irrational ; see fig . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . we believe that most articles published in jcamd have dealt with aspects of drug design left out of fig . the advent of faster computers like first the vax / vms , then supercomputers such as the cray , and finally the pc , have allowed scientist to numerically solve chemical problems of ever increasing size and complexity . in parallel , the development of molecular mechanics force fields combined with the fact that newton s equations of motion could be solved for entire proteins in their aqueous environments were true innovations in the investigation of the structure function relationships [ 2028 ] . the perception that the underlying mechanism of protein ligand recognition would be unravelled and would thus allow what ever since has been called structure - based drug design has never looked so clear and promising as at that particular moment in 1986 . with the exception of a very small fraction of ligands that are purely rigid the values of the torsion angles in ligands are determined by the valence electrons of the atoms . the combined knowledge of the ligand structure ( determined by nmr or x - ray ) , the measured binding affinities , and the spatial overlay of the low energy conformations should then be sufficient to establish a structure activity relationship and pinpoint the spatial organization of the recurrent chemical features correlated with activity ( pharmacophore ) . this paved the way for a series of successes for ligand - based drug design [ e.g. the computational process by which the complementary aspects between a ligand and a receptor binding site can be ascertained has been explored with the design of specifically dedicated docking programmes . the approach was computationally cumbersome due to the need to systematically search all possible ligand orientations within the pocket and scoring each of these poses by its steric hindrance . subsequent developments have taken place in several directions : improved scoring functions [ 5263 ] different ways to deal with ligand flexibility [ 60 , 6472 ] , and most recently also ways to deal with receptor flexibility [ 7378 ] . fundamental research has been performed into directions such as desolvation energies [ 7983 ] , or other aspects of the force fields used for scoring docking poses [ 66 , 78 , 8496 ] . the idea to calculate from first principles all atomic motions occurring in an active enzyme in its aqueous environment has attracted many scientists to computer aided molecular design . starting with the atomic loci obtained from the x - ray structure of en enzyme a series of snapshots describing the trajectory of the enzyme over time could thus be produced and ensemble average properties calculated based on boltzmann s ergodic hypothesis . in silico , one is not bound by the sequential order of events that govern paths between states , and hence so - called thermodynamic cycle methods could be developed that replaced chemical steps with alchemical steps that in principle should lead to the same outcome [ 29 , 30 , 97 , 98 ] . initially , the technique was more a concept than an effective tool as computer power was very limited and molecular descriptors as well as dedicated algorithms needed to be developed . the underlying idea that the 3d dimensional steric / non - bonded ( van der waals ) and electrostatic potential fields generated by the spatial organization of the chemical features around the scaffold of a ligand ( fig . 3 ) play a fundamental role in the biomolecular receptor recognition was so intuitively right and the technique made a break - through in 1988 . about 15% of all articles in jcamd refer to the use of this technique , refined and applied in all sorts of ways to produce the overly famous quantitative structure activity relationship ( qsar ) equations . however , comfa suffers from three drawbacks : ( 1 ) the alignment of the ligands in the pocket must be either known or gambled correctly ; ( 2 ) the method has been established for rigid or quasi - rigid classes of molecules ( e.g. certainly , the best way to apply comfa is to combine it with a pharmacophore model and a carefully conducted conformational study of the ligands .fig . 3contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups many drug design projects include at some stage knowledge of the 3d structure of the target protein , and homology modelling is normally used when neither x - ray nor nmr derived coordinates are available [ 111118 ] . many computer programs were written for this purpose [ 119123 ] and the casp competition illustrates every 2 years where the field stands . similar to the casp competitions , the gpcr - dock [ 126 , 127 ] competitions have evaluated the quality of docking software , but with the additional complexity that the target structures needed to be modelled before docking could be attempted . in recent years a whole series of studies have been published in which homology modelling , combined with other tools , proved a viable replacement for the cumbersome experimental determination of target structures [ 111 , 114 , 128 , 129 ] . the good performance of two dutch teams in the recent gpcr dock competition beautifully illustrates the often mentioned fact that even the best tools only perform well in the hands of good scientists . in the early 1990s the radical new idea emerged that instead of the virtual and/or real screening of large libraries of already existing molecules to identify new bioactive hits , one could rather attempt to construct entirely new synthesizable molecular entities solely based on the knowledge of the active site of the pharmaceutical target enzyme . to do so , small organic fragments composed of few atoms only must be assembled in silico inside the binding sites of enzymes in such a way that optimal protein ligand , steric , and electronic complementarity is achieved [ 84 , 125 , 135141 ] . the major problem of this approach arises from the complexity of the active site landscape and the combinatorial vastness of all possible arrangements of fragments in the volume delineated by an enzyme active site [ 66 , 142144 ] . how to choose the first fragment and where should it be positioned and oriented with respect to the inner surface of the binding pocket or cleft [ 143 , 145 , 146 ] ? the genetic algorithms [ 66 , 142 , 148150 ] have been invented which allow this concept to be realized within a tractable amount of computer time by performing random transformations on a ligand collection . these transformations are selection , mutation , and crossover , and are reminiscent of the corresponding evolutionary processes in biology underlying the optimisation of genes , hence their name genetic algorithms. randomly screening very large libraries containing up to 10 or even 10 chemical entities in in vitro enzymatic assays to produce leads has been the central paradigm of the pharmaceutical industry across the 1990s . however , after years of operating very expensive screening facilities , it has been realized that the hits produced were not of the expected quality . for example , often a bias is observed toward too lipophilic compounds that are impossible to optimize . compared to the actual number of chemically entities ( ~infinite ) the any amount of compounds that can be screened via this process is essentially zero [ 151 , 152 ] . in parallel , computational chemists had inferred that screening could be successfully operated virtually throughout computers at all stages in the drug design process from hit identification via hit optimization to lead optimization [ 153162 ] . thus , libraries of compound that do not actually exist can be screened and a much smaller , manageable number of compounds selected . scaffolds of lead compounds usually carry a number of branching points were chemical variation is allowed . for instance , across a series of chemical substituents sorted by increasing polarity the measured affinity may respond linearly only for a restricted number of them because steric hindrance or global effect such as desolvation may penalize the binding of slightly larger groups . that are insensitive to the spatial alignment of the ligand scaffolds and that are able to recognize particular combinations of properties distributed around the scaffold of a set of active ligands [ 168170 ] . artificial intelligence can be ubiquitously implemented at various stages in the rational drug design process to improve results that can be otherwise be more uncertainly obtained with classical methods , especially when assessing general properties that are the result of the subtle combination of many different factors in relation to others such as drug - likeness . we apologise to the many authors of methods that did nt make it into the above list ( see esm table 1 ) . much good work has been done that the editors certainly would nt allow us to include because citing all 1,200 articles published in jcamd in the first 25 years would perhaps be a bit excessive . the recent work by zhou et al . on the use of dft calculation to accurately assess the existence of intermolecular h - bonds in docking instances . sarmah et al s work on solvent effects also added significantly to the drug - designers toolbox , but the methods described in these articles did nt achieve hype status . the rapid increase in costs of developing and marketing new medicines is not leaving the pharmaceutical industry untouched . recent years have seen a strong concentration of activities in terms of mergers , buy - outs , and closures . it may simply be , that a research - intensive industry like the pharmaceutical industry does not lend itself to the type of management that is common in consumer goods , fashion and footwear . it seems a paradox , though , that the high costs associated with drug design are caused by development , marketing , and legal fees , but when it comes to cost - reduction research departments are , euphemistically called , consolidated . jcamd has published a large series of articles in which multiple methods have been combined . all these pipelines and otherwise combined methods speed up the use of the existing tools , and allow them to be applied to ever larger numbers of small molecules in ever shorter times . actually , there is a new hype raging at the moment , and it is called translational science. in a sense , the recent spate of articles on combining existing techniques into more easily applicable super - tools fit nicely to this translational paradigm . between the lines we read in translational science that the pharmaceutical industry has finally realized that our deep lack of understanding of all aspects of the interaction of a medicine with a human being is the main cause for luck still being the most determining factor in the drug design process . consequently , we see the out - sourcing budgets of the large pharmaceutical industries go up , and more and more fundamental research performed in academia is finding its way to small and medium size enterprises ( smes ) where it can be incorporated in their lean and mean research machines . this new paradigm will probably also be proven a hype soon ; only time can tell if translational research will rescue the pharmaceutical industry , or that it will only better illustrate what it all is that we do nt know yet . it remains a fact that better understanding the underlying biology , better treatment of all available data , and more intelligent combinations of data , information , and knowledge must be beneficial for the drug design process and thus , on the long run , for all of us . if the pharmaceutical industry wants academia more involved in the drug design process they could themselves make a giant first step by making available all ( or at least very many ) x - ray structures of protein ligand complexes . we estimate that the number of pdb [ 189 , 190 ] files collecting computer dust in the pharmaceutical industry is considerably bigger than the 75,000 structures now in the pdb . we offer to set up a database for these files , and we offer to re - refine all industrial structures of protein ligand complexes . we will then only release those coordinates to the wider public that pass certain minimal validation criteria . in pdb_redo we significantly improved 85% of all presently available pdb files that were solved by x - ray . despite massive efforts in the design of tools , databases , robotic techniques , and management innovations , luck seems to be at the basis of the discovery of most new medicines . performed a massive literature search for aryloxypropanolamines and similar compounds binding to the serotonin 5ht-1a receptor and a series of sequence similar amine receptors . a correlation analysis revealed that only one residue s presence / absence showed a perfect correlation with binding / non - binding of a series of compounds . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available in another gpcr related project aimed at using as much heterogeneous data as can possibly be combined , oliveira et al . predicted the role of all active site residues in gpcrs , the pivotal role of arg-340 , and even a series of residue interactions involved in the activation process , and the presence and location of helix viii . the recent flurry of articles on gpcr xray structures [ 206209 ] , and especially the structure with a covalently agonist - bound g protein showed all these predictions to be conceptually right . their systems ( some of which are freely accessible from their website ) revolve around a structure based , and thus very accurate multiple sequence alignment ( msa ) for a whole protein super - family . this msa then functions as the anchor on which to position all kinds of data that can range from 3d structures to genome related data , from mutation studies to ligand binding constants , or from sequence correlation patterns to the prediction of mutations that enhance the protein s stability . a recent development that will aid the drug hunters of the future . showed how this programmable pdf reader could be used to directly couple data in articles on gpcrs to the gpcrdb . first , the residue numbering problem gets solved because the reader can ask the gpcrdb for the position in the gpcr msa of any residue mentioned in the article , and it can even modify or correct the sequence numbers in the article if needed . much good gpcr mutation data was published in the pre - gpcr - structure era that ended with the opsin structure article , and often these data were misinterpreted because of the poor quality of the available homology models . the utopia - gpcr pdf reader can correct those interpretations thereby salvaging old , high quality experimental data for future use . figure 5 shows an image from an old mutation study in which the authors describe several ground - breaking mutations in the guinea pig histamine h1 receptor , building and validating a homology model using these data , and arguing , for example , that residue trp161 plays an important role in receptor - ligand binding . by contrast , the gpcrdb generated annotation listed in the sidebar of the reader indicates that this residue , located in tm iv , points towards the membrane and possibly interacts with cholesterol . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . this analysis required a substantial amount of work : categorizing structures and compounds , creating multiple sequence alignments , analyzing ligand contacts , and transferring the results into a homogeneous residue numbering scheme ( the so - called 3d numbers ) . 6bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . in all these articles the use of a common 3d numbering scheme enables transfer of heterogeneous information between protein family members . the ligands were placed in the same orientation as found in their native pdb file . what if suite if , one day , all structures of nr - ligand complexes that now are scattered over inaccessible industrial hard disks could be concentrated in one system , then we could consider asking much more elaborate questions . project hope aims to predict the molecular phenotype of point mutations that were shown causally related to human disease states . hope only asks the user to cut - n - paste the sequence , and click the residue mutated and the mutation residue type . we believe that the recent spate of consolidations in the pharmaceutical industry is not a problem but an opportunity . mankind needs medicines , and now that pushing ones luck is slowly becoming a less successful technique , only research can find them . this research can progress rapidly if the thousands and thousands of x - ray structures of protein ligand complexes would find their way from hard - disks behind pharmaceutical industry firewalls to the public domain . drug design research in the next 25 years will revolve around ever broader collaborations , ever more holistic understanding of the drug human interactions , and ever better use of the available data , information , and knowledge . supplementary material 1 ( docx 89 kb ) supplementary material 1 ( docx 89 kb )

our current picture of cell nuclear function is shifting from the idea of fully assembled molecular complexes towards molecules freely roaming in nuclear space ( pombo and cook 1996 ; phair and misteli 2000 ; bentley 2002 ) . here , transient interactions ( protein protein , protein dna , and protein rna ) result in a stochastic but directional progression of multi - scale and multi - player processes ( elf et al . 2007 ; zenklusen et al . 2008 ; darzacq et al . 2009 ) . while this change in paradigm challenges our view of the composition of multi - protein complexes , it also raises questions of how the nuclear space is organized , how the availability of factors is regulated and how interaction sites are found by the right interaction partners . while no membrane - bound compartments ( such as in the cytoplasm ) have been identified specific areas can be labeled , including the nucleolus , pcg bodies , nuclear speckles , cajal bodies , gems , cleavage bodies , perinucleolar compartments , the sam68 nuclear body , pml bodies , etc . ( spector 2001 ) forming a patterned space . as a result , besides crowding effects also found in the cytoplasm , molecules in the nucleus encounter a highly complex , anisotropic landscape ( richter et al . 2007 ) . to probe the dynamic functions of this landscape , an ensemble of technologies ( such as frap , flip , confocal imaging etc . ) have been applied to elucidate the dynamics of protein factors and rna particles ( becker et al . many functional protein factors have been found to vary in their immobile versus mobile states , as well as in their average off rates from potential binding sites ( misteli and spector 1999 ; grunwald et al . similarly , a wide range of mobility distributions exist in rna studies ( politz et al . even nuclear bodies , that appear stationary in imaging , are actually dynamic complexes undergoing a steady exchange of their building blocks ( politz et al . these data draw a picture of a highly mobile environment with changing fractions of immobilized populations and a continual change in composition . experimental access to probe this environment and its embedded processes at the molecular scale is limited as : nuclear organization is actively maintained and so can only be studied in living cells ; ensemble technologies , being diffraction limited , fail when challenged by molecular interactions on the nanometer length scale ; and the high dynamics of these often transient interactions demand collection of data in the milliseconds time scale , limiting the achievable signal - to - noise ratios . while multiple strategies are employed today to challenge the resolution criteria ( hell 2003 ; betzig et al . 2006 ) , imaging of single - molecule signals can provide the position of the observed molecule with an accuracy that can overcome the resolution limit by a factor of ten in the living cell , and is in principle capable of providing nanometer precision1 ( bobroff 1986 ; schmidt et al . continuous image acquisition also enables following a single protein or rna over an extended amount of time utilizing the high localization accuracy for tracking rather than construction of a high resolution image . an immediate advantage is that no synchronization of cellular processes is needed as single - molecule tracking ( smt ) is fast enough to observe the sequence of events in real time and synchronization is achieved during data analysis . finally , by providing superb time and spatial information simultaneously , smt is perhaps the tool of choice when it comes to investigating the dynamic landscape of molecular interactions in the living cell . the question however is how fast smt can be , or in other words , what is the fastest process observable by this technology ? smt has been shown to be fast enough to even follow freely moving molecules in an aqueous solution ( grunwald et al . measurements of nucleocytoplasmic transport have shown definitively that transient interactions in the living cell can be resolved on the nanometer length- and milliseconds time scales using smt . in the same way , single protein factors and rnas in the nucleus have been measured using inert test probes and functional protein factors ( goulian and simon 2000 ; kubitscheck et al . compared with fluorescence correlation spectroscopy ( fcs ) or fluorescence recovery after photobleaching ( frap ) , smt provides the means to analyze multiple forms of mobility in heterogeneous environments . even more , smt enables observation of the molecular behavior over the whole field of view and not only within either the confocal volume of an fcs spot or the bleached area in frap . this makes the technique very valuable for studying intranuclear processes of proteins and rnas in relation to defined nuclear structures . however , observation of single molecules in living cells needs to overcome the problem of having too many overlapping signals from the labeled population of molecules . adjustment of the number of labeled molecules is usually done by : providing small amounts of recombinant fluorescently labeled proteins ( e.g. , by microinjection ) , by careful titration of labeled ligand concentrations , by adjusting expression levels of fluorescent reporter molecules ( e.g. , using leaky promoters ) , or by signal amplification ( e.g. , dna probes , molecular beacons or the ms2 system ) ( bertrand et al . the resulting experimental design is then driven by optimizing the signal - to - noise ratio during data acquisition , usually by a combination of background reduction , signal multiplication on the detector ( e.g. , apds or emccds ) and very sensitive detection at low read noise conditions . finally , the observation time needs to be maximized , taking photobleaching rates , desirable signal intensity and applicable amounts of light into consideration . while many pioneering studies used excitation energies in the range of several kilo watts per square centimeter , cells and especially nuclear structures and processes are sensitive to high - photon fluxes ( dobrucki et al . 2007 ; 2009 ) . in this context , calibrating the amount of light delivered to the cell is of crucial importance and can be done for broadband light sources as well as for laser based imaging ( grunwald et al . 2008b ) . depending on the mobility of the observed molecules and the acquisition speed of the microscope system used , the acquired data is then analyzed based on the tracking of individual signals ( see ( grunwald et al . 2008c ) for a detailed review ) . figure 1 details simple modifications that can be applied to commercially available equipment to improve performance for the detection of single molecules in living cells at appropriate time scales . in short , a laser illumination module ( here shown in a customized home - built version , but also commercially available ) is combined with a fast switching mechanism and a fast intensity regulator for the laser light and fiber coupled to the microscope . a stable stage with high precision and small drift is used to position samples above the objective lens and to allow for cell manipulation ( e.g. , microinjection ) if needed . lastly , a sensitive , fast camera , typically an emccd , is used for recording the single - molecule signal and is mounted as directly as possible onto the microscope to increase photon transmission . either sequential imaging or a second detector is used to monitor a cellular reference structure . a conventional wide - field epifluorescence microscope is custom - modified to optimize single molecule detection in biological samples . the excitation ( laser ) light is merged using dichromatic beam splitters and mirrors and coupled into an optical mono - mode fiber to deliver the light to the microscope . an acousto - optical tunable filter is used to attenuate laser power and switch illumination on and off with microsecond resolution . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , a microinjection system allows delivery of fluorescently labeled probes into a living cell an smt microscope setup . a conventional wide - field epifluorescence microscope is custom - modified to optimize single molecule detection in biological samples . single - mode , low - noise lasers are the preferred choice . the excitation ( laser ) light is merged using dichromatic beam splitters and mirrors and coupled into an optical mono - mode fiber to deliver the light to the microscope . an acousto - optical tunable filter is used to attenuate laser power and switch illumination on and off with microsecond resolution . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , is used to detect reference images . a microinjection system allows delivery of fluorescently labeled probes into a living cell to summarize , smt and fcs provide the means to study single molecules or very small , locally resolved , ensembles of fluorescently labeled molecules in the living cell . in the following , we discuss our current view of the nuclear landscape , dynamical aspects of the nucleus and point out controversial areas that remain . the road to live cell imaging was laid down by confocal microscopy and today s fcs implementations are building upon this technology ( eigen and rigler 1994 ) . the first experiment to provide a mobility profile of the nuclear space used fcs and fluorescein - labeled oligodeoxynucleotides ( oligos ) to measure intranuclear diffusion coefficients in living cells ( politz et al . 1998 ) . labeled poly - thymidine oligos , that are passively taken up by living cells , formed hybrids with the poly - adenylated tails of rnas ( poly(a)rnas ) in the nucleus , and two major fractions of diffusion rates were observed . besides the free oligonucleotide diffusion , a slow moving fraction , interpreted as the labeled diffusive rnas ( d = 10 m / s ) , was detected . a third minor fraction was also detected with a 10-fold slower mobility rate ( see table 1 ) . in another experiment , the effect of nuclear compartments on the movement of poly(a)rnas in the nucleoplasm was compared to poly(a)rna mobility in speckles ( politz et al . these values are similar to the mobility of the slower class reported in the first study ( table 1 ) . interestingly , temperature reduction from 37c to 22c had no detectable effect on poly(a)rna mobility , and was interpreted as meaning that movement between the nucleoplasm and speckles does not require metabolic energy , which is in stark contrast to a previous report ( calapez et al . table 1overview of nuclear diffusion coefficients measured by smt and fcsreferenceconstructmethoddiffusion coefficientsubcompartment(grunwald et al . 2006b)u1 snrnp bioactivesmtd = 0.5 to 8 m / snucleoplasm , speckles(grunwald et al . 2008a)streptavidin - nls inertsmtd = 0.8 to 5 m / snucleoplasm , nucleolus , heterochromatin(speil and kubitscheck 2010)ovalbumin inertsmtd = 0.5 to 12 m / snucleoplasm , nucleolus(bancaud et al . 2009)gfp - monomerfcsd1 = 29 m / seuchromatingfp - monomerd1 = 87 m / saqueous solutiongfp - dimerd2 = 17 m / seuchromatingfp - dimerd2 = 55 m / saqueous solutiongfp - pentamerd3 = 7.7 m / snucleoplasm(politz et al . 1998)oligo(dt):poly(a ) rnafcsd = 1 to 10 m / snucleoplasm(politz et al . 2006)oligo(dt):poly(a ) rnafcsd1 = 0.65specklesd2 = 0.7nucleoplasm(shav - tal et al . 2004)yfp - ms2 mrnpfrapd = 0.09 m / snucleoplasmsmtd = 0.04 m / snucleoplasm(van den bogaard and tyagi 2009)gfp - mrna-96-mersmtd = 0.033 m / snucleus(siebrasse et al . 2008)dna - labeled br mrnpsmtd = 0.24 to 4.0 m / ssalivary - gland cell nucleus(mor et al . 2005)hiv - infcsd = 10.5 m / snucleusin these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . for details , see original literature overview of nuclear diffusion coefficients measured by smt and fcs in these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . for details , see original literature probing the nuclear space with inert probe molecules , initially described by grunwald et al . 2008a using smt , was then used to explore the effects of molecular crowding on diffusion and binding of nuclear proteins in heterochromatin using fcs technology ( bancaud et al . volume exclusion , diffusive barriers in more dense nuclear compartments and transient trapping in heterochromatin , as observed by grunwald et al . photoactivation experiments were carried out , and in euchromatin , diffusion and binding parameters were well explained by a diffusion limited model . however , when the same probes were tested in heterochromatin , where slower kinetics were expected ( dependent on the heterochromatin to euchromatin abundance ratio ) , an unexpected biphasic mobility was observed that could not be explained by a diffusion reaction model or a random crowding model ( bancaud et al . based on these results the authors deduced fractal geometry of chromatin as previously suggested ( wachsmuth et al . circumventing the small active measurement area and time averaging inherent to fcs , improvements in camera technology , and the development of rna labeling systems , made imaging of messenger ribonucleoprotein particles ( mrnps ) in living cells a possibility . one such labeling system is the ms2 system that consists of two components , i.e. , a sequence of ms2 rna stem - loops introduced into the rna sequence of interest and a coat protein to which a fluorescent protein is fused . the coat protein binds to the rna sequence with nanomolar affinity thereby generating a multiplexed signal sufficient for single molecule tracking ( stockley et al . lago et al . 2001 ) . to detect mrnps in real time in living mammalian cells , 24 ms2 stem - loops were inserted downstream of a target rna ( shav - tal et al . movements of individual mrnps were followed for 100 frames using exposure times of 250 and 333 ms , respectively . the tracking criteria for mrnps was set to more than eight frames of continuous observation , and the mean diffusion coefficient , in stark contrast to the fcs data cited above , was found to be 0.04 m / s at 37c . this diffusion was also temperature dependent , as at room temperature the diffusion coefficient was reduced indicating that mrnps released after transcription moved probabilistically through the nucleoplasm following simple laws of diffusion ( shav - tal et al . when very large mrnps were tracked in the nucleus ( dys - mrnp , ranging from 4.8 to 14 kb ) , an even lower mobility of mrnps was found ( d = 0.005 m / s ) ( mor et al . 2010 ) . interestingly , in this report , mrnps were found to travel in chromatin free zones in a channeled diffusion manner with the accumulation of mobile particles in distinct chromatin free zones ( mor et al . 2010 ) . while there is evidence of open spaces in the lamin network as revealed by three - dimensional structured illumination microscopy ( 3d - sim ) ( schermelleh et al . 2008 ) , it is unclear whether these spaces are indeed channels and if they exist over the entire nucleus as predicted ( mor et al . 2010 ) . another labeling approach uses molecular beacons for fluorescent labeling of mrnp ( van den bogaard and tyagi 2009 ) . a molecular beacon is composed of a stem hybrid that keeps the fluorophore close to a quencher rendering the probe initially non - fluorescent . when the probe hybridizes to a target sequence , the fluorophore separates from the quencher due to a conformational change , and becomes detectable . sequence specific multiplexing of probes on a target rna is possible , reaching up to 100 probe copies / rna , resulting in signal intensities of single mrnps that can be detected as diffraction - limited spots in living cells . in an experiment where imaging was performed at 300 ms integration time , an average diffusion coefficient of 0.033 m / s was found for one half of the observed mrnps , while the other half displayed a stationary behavior with a calculated diffusion coefficient of 0.0006 m / s . when the single mrnps positions were mapped to a chromatin density image the stationary particles were located in high - density chromatin regions . atp depletion increased the number of stalled particles but no strong effect on diffusion was found , again suggesting that mrnp movement is an atp - independent process ( van den bogaard and tyagi 2009 ) . all of these results are 10100-fold lower in their diffusion constants ( see table 1 ) compared to the fcs based mobility profile ( politz et al . in contrast to the above studies , however , when an insect model system ( instead of a mammalian cell system ) was used , i.e. , balbiani ring ( br2 ) mrnas ( extremely large mrnas found in the salivary glands of the chironomus midge ) , four different mobile fractions , widely varying , were found approaching diffusion coefficients comparable to those found by fcs ( table 1 ) ( siebrasse et al . interestingly , the distribution of diffusion coefficients is better explained as a probability distribution of mobile states that an mrna molecule can adopt , rather than by the existence of distinct classes of differently mobile mrnas , a first indication of which was already seen in protein mobility ( see fig . 2 and ( grunwald et al . 2008a ) ) . fluorescent labeling here was done either by microinjection of oligos complementary to br2 mrna or with a labeled - hrp36 protein that specifically binds br2 mrna ( siebrasse et al . 2nuclear protein mobility as determined by smt . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . jump distances were binned into three groups of 080 nm , 80240 nm , and larger than 240 nm displacement . no difference in observation frequency and decay time is observable between the three binned classes . b the same three classes are used to analyze jump distances of streptavidin molecules inside mecp2-induced heterochromatin . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . d time projection of the trace analyzed in c projected onto the reference image from the cell nucleus . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) nuclear protein mobility as determined by smt . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . jump distances were binned into three groups of 080 nm , 80240 nm , and larger than 240 nm displacement . no difference in observation frequency and decay time is observable between the three binned classes . b the same three classes are used to analyze jump distances of streptavidin molecules inside mecp2-induced heterochromatin . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . d time projection of the trace analyzed in c projected onto the reference image from the cell nucleus . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) as there are such dramatic differences between the fcs data and tracking data of one group versus another , it is very clear that controversy exists in this field , which might be explained by clear technical differences in how the experiments were performed . as we have seen above that rna tracking is possible , it would be interesting to also track single proteins as proteins are the facilitators of many major cellular processes . one such study looked at the mobility of fluorescently labeled uridine - rich small nuclear ribonucleoproteins ( u1 snrnps ) , biologically active splicing factors ( grunwald et al . gfp - labeled asf / sf2 was used to mark nuclear speckles allowing direct comparison of u1 snrnp dynamics inside and outside of the nuclear speckles . using high speed fluorescence microscopy , with frame rates of up to 200 hz , no significant mobility was found for 80% of u1 snrnps , possibly caused by molecular trapping in nuclear structures as well as immobilization in spliceosomes and post - splicing processes . a continuous range of mobility for u1 snrnps , ranging from 0.5 to 8 m / s was found . the diffusion coefficient of 0.5 m / s corresponds to impeded uncomplexed single u1 snrnps or higher organized spliceosome - complexes . correspondingly , using frap experiments , a three to five fold reduction of the diffusion coefficient of larger molecules in the nuclei was also found ( gorski et al . from here we conclude that there is not just one kinetic condition for association and dissociation events of biologically active proteins in the nucleus . how does the large immobile fraction of u1 snrnps compare , however , to inert proteins ? tagged streptavidin coupled to a nuclear localization sequence ( nls ) with a size of about 60 kda , and a second probe , ovalbumin with a size of 45 kda , were tracked in living cell nuclei ( grunwald et al . while streptavidin is not translocated by nuclear pores , primarily due to its size , ovalbumin likely passively transports into the nucleus . using high speed fluorescence imaging at frame rates of up to 200 hz , the streptavidin experiment succeeded in capturing even single traces of probe molecules and deduced a diffusion rate comparable to that of the inert gfp protein as seen by fcs ( see table 1 and fig . 2 ) ( grunwald et al . 2008a ; bancaud et al . 2009 ) . different nuclear compartments affect the movement of inert proteins differently , but even in the nucleoplasm , two kinetic components ( mobile and trapped ) were observed , and the mobile fraction is widely spread over a wide range of diffusion coefficients ( the data could not be fitted satisfactorily assuming only one or two diffusing components , table 1 ) . compared with the nucleoplasm ( defined as space neither labeled by mecp2 or asf1 exclusion ) , proteins seemed to become trapped predominantly in pericentric heterochromatin resulting in fewer proteins moving freely ( see fig . 2 ) . even more exciting , and in contrast with the fcs study discussed above ( bancaud et al . 2009 ) , proteins were trapped less frequently in the nucleolus compared to the nucleoplasm . while confocal imaging implies that the nucleolus excludes the test protein , the mobility data suggest minimal trapping in this compartment and combined with no retention of proteins at the compartment interface , exclusion turns out to be a dynamic effect , where mobile proteins leave the nucleolus frequently and fast ( grunwald et al . 2008a ; speil and kubitscheck 2010 ) . when ovalbumin was used , an inert protein that does not contain an nls , comparable observations were made demonstrating the limited , if any , impact of the sv40 nls - related electric charge on protein mobility in the nucleus . an important question that can be addressed using single molecule tracking is the mobility of hiv ( human immunodeficiency virus ) related factors , their interaction times and regulation of cellular distribution . we first provide a brief summary of the hiv lifecycle , including import and export processes of hiv genetic material through the nuclear membrane , which is a very elegant symphony between cytoplasmic and nuclear viral and host cell factors . single molecule tracking techniques might enable deeper understanding of the spatial - temporal dynamics of these processes thereby not only providing key information on very basic biological processes , but we surmise , also exposing vulnerabilities that could be targeted in the fight against this devastating disease . the hiv virus is an rna lentivirus with a genome comprising two single - rna strands containing the full genetic information needed for viral replication in host cells ( luciw et al . 1996 ; fields et al . 2007 ; levy 2007 ) . through the process of fusion and endocytosis 3 ) , by binding its trimeric surface gp120 to a cd4 molecule present on the host cell ( reviewed in ( gallo et al . 2003 ) ) . this binding induces a conformational change to a specific chemokine receptor ( predominantly ccr5 or cxcr4 ( berger et al . 1999 ) ) , and this in turn induces a conformational change in gp41 , another viral surface factor . this fusion of the viral and infected cell s membranes results in the release of the hiv viral core into the cytoplasm ( chan et al . 2010 ) and the viral reverse transcription complex , comprising viral and host proteins , is assembled and cdna synthesis begins . the result of reverse transcription is an hiv preintegration complex ( pic ) , which is then challenged with importing itself into the nucleus . it is still unclear how the pic imports into the nucleus ( dvorin et al . 2002 ; bukrinsky 2004 ; yamashita and emerman 2005 ; yamashita and emerman 2006 ) . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 5 while fully spliced rnas are exported as usual to the cytoplasm , singly spliced or unspliced rnas are also exported through the nuclear pore ( 6 ) via a rev - mediated mechanism , again assisted by host proteins . 7 packaging of new virions occurs at the host cell s membrane . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt the life cycle of hiv . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 5 while fully spliced rnas are exported as usual to the cytoplasm , singly spliced or unspliced rnas are also exported through the nuclear pore ( 6 ) via a rev - mediated mechanism , again assisted by host proteins . 7 packaging of new virions occurs at the host cell s membrane . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt following transport across the nuclear pore complex viral and host proteins again participate in integration of the viral genome into the host cell genome ( greene and peterlin 2002 ) . while this is primarily mediated by integrase ( in ) , which binds to the ends of the viral dna , host proteins are also involved and required , though their precise functions remain unknown ( kalpana et al . 1994 ; lee and craigie 1994 ; farnet and bushman 1997 ; li et al . 2000 ; suzuki and craigie 2002 ; beitzel and bushman 2003 ; lin and engelman 2003 ) . ledgf / p75 also associates with in and has also been implicated in participating in its nuclear import and/or the integration process ( cherepanov et al . 2003 ; maertens et al . 2003 ) . as the hiv provirus can integrate into many different chromosomal locations in the cell it is tempting to explain viral latency ( vs. transcriptional activity ) as integration into repressed heterochromatin . while this is at least in part true , it is not the whole story ( for reviews on hiv viral latency , see ( han et al . 2007 ; coiras et al . 2009 ; graci et al . 2009 ) ) . in the case of a transcriptionally active integrated provirus , the 5 ltr ( long terminal repeat containing promoter elements ( taube et al . 1999 ) ) positions rna polymerase ii ( rnapii ) at the site of initiation of transcription and is responsible for the assembly of the pre - initiation complex . while transcription can begin with these minimal components , rnapii invariably fails to elongate efficiently ( kao et al . is required which associates with host cyclin t1 , which in turn recruits host cdk9 . tat binds the 5 bulge region of tar ( a 59-nucleotide stem - loop rna element in the ltr ) via its arginine - rich motif and recruitment of p - tef - b ( the complex formed by cyclin t and cdk9 ) results in hyper - phosphorylation of the c - terminal domain of rna polymerase ii , thereby stimulating efficient transcriptional elongation ( zhou et al . the integrated provirus , while only 9 kb long , then successfully expresses 15 distinct viral proteins , facilitated by an elegant and complex splicing mechanism that involves both complete and incomplete splicing ( frankel and young 1998 ) . when the mrna is completely spliced ( encoding for nef , tat , and rev ) it is rapidly transported into the cytoplasm and transcribed ( cullen 1998 ) . when the mrna , however , is singly spliced or unspliced , viral transcripts remain in the nucleus and are relatively stable ( luo et al . 1994 ; powell et al . 1997 ) . while the export of unspliced ( or partially spliced ) rna in eukaryotic cells is usually prohibited , the virus overcomes this block by utilizing the viral protein rev ( custodio et al . the rev protein binds to an rev response element ( rre ) encoded in the viral rna sequence and together with recruited host proteins manages to export the unspliced or partially spliced rna in what is referred to as the rev - rre - crm1 export mechanism ( reviewed in ( hope 1999 ) ) . while the partially ( or singly ) spliced viral transcripts encode the structural enzymatic accessory proteins , the unspliced rna species constitute the genome of newly formed progeny virions , and export of these rnas depend heavily on rev s leucine - rich nes ( nuclear - export sequence ) as well as on host proteins , predominantly ran gtpase ( fornerod et al . 1997 ) . as the viral components needed for new virion formation are assembled in the cytoplasm , shuttling to the plasma membrane ( where budding will occur ) takes place ( reviewed in ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) ) . this process is highly reliant on the viral gag polyprotein , but involves , once again , a number of host cell factors , without which the process does not occur ( dussupt et al . the terminal step in the budding reaction involves a second membrane fusion event for the virus , and this too is an orchestrated effort between the viral gag polyprotein and host proteins ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) . the virion is then released and free to continue the vicious cycle of infection . where to from here ? as becomes evident from the wealth of information already available , the past several decades of research have defined a large number of host cell proteins that influence every step of the viral life cycle ( ptak et al . 2008 ; fu et al . 2009 ; pinney et al . 2009 ) . initial imaging results on the localization of , for example cd4 and ccr5 on the cellular membrane ( steffens and hope 2003 ; steffens and hope 2004 ) , rna distribution in virions ( chen et al . 2009 ) , and the biogenesis of hiv virions at the plasma membrane ( jouvenet et al . however , a report that investigated in mobility within the nucleus by fcs , also demonstrates the current limitations at the nanometer length- and milliseconds time - scale ( maertens et al . clear examples of how smt could be applied to the study of hiv include analysis of the import and export of viral rna and proteins , e.g. the pic complex , the interaction durations and sites for the rev - rre - crm1 complex , the distribution of genome integration sites or the interplay of tat and rnapii . while the above are good examples of what can be achieved with single - color smt , investigating the interaction times and complex formation of , for instance hiv - rna and rev or in protein , will require simultaneous multi - color smt . the problem with this approach is that while in each individual color single molecules will be localized with high precision , the colocalization of these single - molecule positions will remain diffraction limited , hence in the range of more than 200 nm . while single - molecule motors , imaged directly on the cover glass in solution have been double labeled and registered with sub - diffraction - limited precision ( churchman et al . 2005 ) , this virtually renders multi - color smt in the living cell impossible , as in the living cell specific problems with aberrations and chromatic effects inherent to the cellular environment need to be overcome . this problem appears to be overcome by the recent development of a super - registration technique for single molecules with high spatial precision between spectral channels . using a nuclear pore fluorescent stain it was possible to achieve a 10-nm local registration of nuclear pores and endogenous mrna , labeled with the ms2 system . super - registration allowed following interactions of single mrnas and pores during export and resolving individual transient steps of the export process and their respective rate constants , which were previously undefined ( grunwald and singer 2010 ) . in conclusion , the hiv life cycle demonstrates how important it is to gather a complete picture in time and space of cellular and viral mechanisms that , without microscopy , might not be possible . the horizon for the future should include single - molecule studies on the virus dynamic equilibrium of processes in living cells that , until now , have been out of reach . even more , the perspective of investigating individual complexes and molecular interactions in the living cell at the single - molecule level , e.g. , through developments like super - registration , guides the way towards a quantitative picture of the dynamic equilibrium of cellular life .

cellular life can be described as a dynamic equilibrium of a highly complex network of interacting molecules . for this reason , it is no longer sufficient to only know the identity of the participants in a cellular process , but questions such as where , when , and for how long also have to be addressed to understand the mechanism being investigated . additionally , ensemble measurements may not sufficiently describe individual steps of molecular mobility , spatial - temporal resolution , kinetic parameters , and geographical mapping . it is vital to investigate where individual steps exactly occur to enhance our understanding of the living cell . the nucleus , home too many highly complex multi - order processes , such as replication , transcription , splicing , etc . , provides a complicated , heterogeneous landscape . its dynamics were studied to a new level of detail by fluorescence correlation spectroscopy ( fcs ) . single - molecule tracking , while still in its infancy in cell biology , is becoming a more and more attractive method to deduce key elements of this organelle . here we discuss the potential of tracking single rnas and proteins in the nucleus . their dynamics , localization , and interaction rates will be vital to our understanding of cellular life . to demonstrate this , we provide a review of the hiv life cycle , which is an extremely elegant balance of nuclear and cytoplasmic functions and provides an opportunity to study mechanisms deeply integrated within the structure of the nucleus . in summary , we aim to present a specific , dynamic view of nuclear cellular life based on single molecule and fcs data and provide a prospective for the future .

Introduction Observing single molecules in the nucleus, a possibility? Nuclear mobility as seen by fluorescence correlation spectroscopy Single RNA tracking in the nucleus Single protein tracking in the nucleus Imaging HIV in the living cella perspective The life cycle of HIV

here , transient interactions ( protein protein , protein dna , and protein rna ) result in a stochastic but directional progression of multi - scale and multi - player processes ( elf et al . while this change in paradigm challenges our view of the composition of multi - protein complexes , it also raises questions of how the nuclear space is organized , how the availability of factors is regulated and how interaction sites are found by the right interaction partners . while no membrane - bound compartments ( such as in the cytoplasm ) have been identified specific areas can be labeled , including the nucleolus , pcg bodies , nuclear speckles , cajal bodies , gems , cleavage bodies , perinucleolar compartments , the sam68 nuclear body , pml bodies , etc . as a result , besides crowding effects also found in the cytoplasm , molecules in the nucleus encounter a highly complex , anisotropic landscape ( richter et al . to probe the dynamic functions of this landscape , an ensemble of technologies ( such as frap , flip , confocal imaging etc . ) these data draw a picture of a highly mobile environment with changing fractions of immobilized populations and a continual change in composition . 2006 ) , imaging of single - molecule signals can provide the position of the observed molecule with an accuracy that can overcome the resolution limit by a factor of ten in the living cell , and is in principle capable of providing nanometer precision1 ( bobroff 1986 ; schmidt et al . an immediate advantage is that no synchronization of cellular processes is needed as single - molecule tracking ( smt ) is fast enough to observe the sequence of events in real time and synchronization is achieved during data analysis . finally , by providing superb time and spatial information simultaneously , smt is perhaps the tool of choice when it comes to investigating the dynamic landscape of molecular interactions in the living cell . measurements of nucleocytoplasmic transport have shown definitively that transient interactions in the living cell can be resolved on the nanometer length- and milliseconds time scales using smt . in the same way , single protein factors and rnas in the nucleus have been measured using inert test probes and functional protein factors ( goulian and simon 2000 ; kubitscheck et al . compared with fluorescence correlation spectroscopy ( fcs ) or fluorescence recovery after photobleaching ( frap ) , smt provides the means to analyze multiple forms of mobility in heterogeneous environments . adjustment of the number of labeled molecules is usually done by : providing small amounts of recombinant fluorescently labeled proteins ( e.g. finally , the observation time needs to be maximized , taking photobleaching rates , desirable signal intensity and applicable amounts of light into consideration . depending on the mobility of the observed molecules and the acquisition speed of the microscope system used , the acquired data is then analyzed based on the tracking of individual signals ( see ( grunwald et al . figure 1 details simple modifications that can be applied to commercially available equipment to improve performance for the detection of single molecules in living cells at appropriate time scales . in short , a laser illumination module ( here shown in a customized home - built version , but also commercially available ) is combined with a fast switching mechanism and a fast intensity regulator for the laser light and fiber coupled to the microscope . lastly , a sensitive , fast camera , typically an emccd , is used for recording the single - molecule signal and is mounted as directly as possible onto the microscope to increase photon transmission . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , a microinjection system allows delivery of fluorescently labeled probes into a living cell an smt microscope setup . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , is used to detect reference images . a microinjection system allows delivery of fluorescently labeled probes into a living cell to summarize , smt and fcs provide the means to study single molecules or very small , locally resolved , ensembles of fluorescently labeled molecules in the living cell . in the following , we discuss our current view of the nuclear landscape , dynamical aspects of the nucleus and point out controversial areas that remain . the first experiment to provide a mobility profile of the nuclear space used fcs and fluorescein - labeled oligodeoxynucleotides ( oligos ) to measure intranuclear diffusion coefficients in living cells ( politz et al . labeled poly - thymidine oligos , that are passively taken up by living cells , formed hybrids with the poly - adenylated tails of rnas ( poly(a)rnas ) in the nucleus , and two major fractions of diffusion rates were observed . in another experiment , the effect of nuclear compartments on the movement of poly(a)rnas in the nucleoplasm was compared to poly(a)rna mobility in speckles ( politz et al . these values are similar to the mobility of the slower class reported in the first study ( table 1 ) . interestingly , temperature reduction from 37c to 22c had no detectable effect on poly(a)rna mobility , and was interpreted as meaning that movement between the nucleoplasm and speckles does not require metabolic energy , which is in stark contrast to a previous report ( calapez et al . for details , see original literature overview of nuclear diffusion coefficients measured by smt and fcs in these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . 2008a using smt , was then used to explore the effects of molecular crowding on diffusion and binding of nuclear proteins in heterochromatin using fcs technology ( bancaud et al . the coat protein binds to the rna sequence with nanomolar affinity thereby generating a multiplexed signal sufficient for single molecule tracking ( stockley et al . the tracking criteria for mrnps was set to more than eight frames of continuous observation , and the mean diffusion coefficient , in stark contrast to the fcs data cited above , was found to be 0.04 m / s at 37c . when very large mrnps were tracked in the nucleus ( dys - mrnp , ranging from 4.8 to 14 kb ) , an even lower mobility of mrnps was found ( d = 0.005 m / s ) ( mor et al . 2008 ) , it is unclear whether these spaces are indeed channels and if they exist over the entire nucleus as predicted ( mor et al . a molecular beacon is composed of a stem hybrid that keeps the fluorophore close to a quencher rendering the probe initially non - fluorescent . when the probe hybridizes to a target sequence , the fluorophore separates from the quencher due to a conformational change , and becomes detectable . in an experiment where imaging was performed at 300 ms integration time , an average diffusion coefficient of 0.033 m / s was found for one half of the observed mrnps , while the other half displayed a stationary behavior with a calculated diffusion coefficient of 0.0006 m / s . in contrast to the above studies , however , when an insect model system ( instead of a mammalian cell system ) was used , i.e. , balbiani ring ( br2 ) mrnas ( extremely large mrnas found in the salivary glands of the chironomus midge ) , four different mobile fractions , widely varying , were found approaching diffusion coefficients comparable to those found by fcs ( table 1 ) ( siebrasse et al . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) as there are such dramatic differences between the fcs data and tracking data of one group versus another , it is very clear that controversy exists in this field , which might be explained by clear technical differences in how the experiments were performed . correspondingly , using frap experiments , a three to five fold reduction of the diffusion coefficient of larger molecules in the nuclei was also found ( gorski et al . from here we conclude that there is not just one kinetic condition for association and dissociation events of biologically active proteins in the nucleus . tagged streptavidin coupled to a nuclear localization sequence ( nls ) with a size of about 60 kda , and a second probe , ovalbumin with a size of 45 kda , were tracked in living cell nuclei ( grunwald et al . different nuclear compartments affect the movement of inert proteins differently , but even in the nucleoplasm , two kinetic components ( mobile and trapped ) were observed , and the mobile fraction is widely spread over a wide range of diffusion coefficients ( the data could not be fitted satisfactorily assuming only one or two diffusing components , table 1 ) . while confocal imaging implies that the nucleolus excludes the test protein , the mobility data suggest minimal trapping in this compartment and combined with no retention of proteins at the compartment interface , exclusion turns out to be a dynamic effect , where mobile proteins leave the nucleolus frequently and fast ( grunwald et al . when ovalbumin was used , an inert protein that does not contain an nls , comparable observations were made demonstrating the limited , if any , impact of the sv40 nls - related electric charge on protein mobility in the nucleus . an important question that can be addressed using single molecule tracking is the mobility of hiv ( human immunodeficiency virus ) related factors , their interaction times and regulation of cellular distribution . we first provide a brief summary of the hiv lifecycle , including import and export processes of hiv genetic material through the nuclear membrane , which is a very elegant symphony between cytoplasmic and nuclear viral and host cell factors . single molecule tracking techniques might enable deeper understanding of the spatial - temporal dynamics of these processes thereby not only providing key information on very basic biological processes , but we surmise , also exposing vulnerabilities that could be targeted in the fight against this devastating disease . the hiv virus is an rna lentivirus with a genome comprising two single - rna strands containing the full genetic information needed for viral replication in host cells ( luciw et al . this binding induces a conformational change to a specific chemokine receptor ( predominantly ccr5 or cxcr4 ( berger et al . this fusion of the viral and infected cell s membranes results in the release of the hiv viral core into the cytoplasm ( chan et al . the result of reverse transcription is an hiv preintegration complex ( pic ) , which is then challenged with importing itself into the nucleus . it is still unclear how the pic imports into the nucleus ( dvorin et al . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt the life cycle of hiv . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt following transport across the nuclear pore complex viral and host proteins again participate in integration of the viral genome into the host cell genome ( greene and peterlin 2002 ) . while this is primarily mediated by integrase ( in ) , which binds to the ends of the viral dna , host proteins are also involved and required , though their precise functions remain unknown ( kalpana et al . as the hiv provirus can integrate into many different chromosomal locations in the cell it is tempting to explain viral latency ( vs. transcriptional activity ) as integration into repressed heterochromatin . while this is at least in part true , it is not the whole story ( for reviews on hiv viral latency , see ( han et al . in the case of a transcriptionally active integrated provirus , the 5 ltr ( long terminal repeat containing promoter elements ( taube et al . 1999 ) ) positions rna polymerase ii ( rnapii ) at the site of initiation of transcription and is responsible for the assembly of the pre - initiation complex . tat binds the 5 bulge region of tar ( a 59-nucleotide stem - loop rna element in the ltr ) via its arginine - rich motif and recruitment of p - tef - b ( the complex formed by cyclin t and cdk9 ) results in hyper - phosphorylation of the c - terminal domain of rna polymerase ii , thereby stimulating efficient transcriptional elongation ( zhou et al . when the mrna is completely spliced ( encoding for nef , tat , and rev ) it is rapidly transported into the cytoplasm and transcribed ( cullen 1998 ) . when the mrna , however , is singly spliced or unspliced , viral transcripts remain in the nucleus and are relatively stable ( luo et al . the terminal step in the budding reaction involves a second membrane fusion event for the virus , and this too is an orchestrated effort between the viral gag polyprotein and host proteins ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) . as becomes evident from the wealth of information already available , the past several decades of research have defined a large number of host cell proteins that influence every step of the viral life cycle ( ptak et al . however , a report that investigated in mobility within the nucleus by fcs , also demonstrates the current limitations at the nanometer length- and milliseconds time - scale ( maertens et al . clear examples of how smt could be applied to the study of hiv include analysis of the import and export of viral rna and proteins , e.g. while the above are good examples of what can be achieved with single - color smt , investigating the interaction times and complex formation of , for instance hiv - rna and rev or in protein , will require simultaneous multi - color smt . the problem with this approach is that while in each individual color single molecules will be localized with high precision , the colocalization of these single - molecule positions will remain diffraction limited , hence in the range of more than 200 nm . while single - molecule motors , imaged directly on the cover glass in solution have been double labeled and registered with sub - diffraction - limited precision ( churchman et al . 2005 ) , this virtually renders multi - color smt in the living cell impossible , as in the living cell specific problems with aberrations and chromatic effects inherent to the cellular environment need to be overcome . this problem appears to be overcome by the recent development of a super - registration technique for single molecules with high spatial precision between spectral channels . using a nuclear pore fluorescent stain it was possible to achieve a 10-nm local registration of nuclear pores and endogenous mrna , labeled with the ms2 system . super - registration allowed following interactions of single mrnas and pores during export and resolving individual transient steps of the export process and their respective rate constants , which were previously undefined ( grunwald and singer 2010 ) . in conclusion , the hiv life cycle demonstrates how important it is to gather a complete picture in time and space of cellular and viral mechanisms that , without microscopy , might not be possible . the horizon for the future should include single - molecule studies on the virus dynamic equilibrium of processes in living cells that , until now , have been out of reach . even more , the perspective of investigating individual complexes and molecular interactions in the living cell at the single - molecule level , e.g. , through developments like super - registration , guides the way towards a quantitative picture of the dynamic equilibrium of cellular life .

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"INTRODUCTION\nMATERIALS AND METHODS\nMedical study\nRESULTS AND DISCUSSION\nMedical study\nPulmonar(...TRUNCATED)

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Introduction Materials and Methods Results and Discussion Conclusions Data Sharing

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1. Introduction 2. Materials and Methods 3. Results 4. Discussion and Conclusions

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