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{"CAPTION FIG5.png": "'Figure 5.: **Clathrin structures containing VSV recruit more cortactin that pits that internalize Di-T.** (A) Cortactin recruitment duringcoated pit formation. Left, snapshot showing the surface of a BSC cell transiently expressing cortactin-EGFP (green) and mCherry-LCa (red) at 18 h post-transfection. Time-lapse images were acquired at 3 s intervals, and frame B3 is shown. Middle, split channel lumpfangsuits of coated pit formation in the cell at left. White arrowheads highlight pits in which cortactin recruitment is clearly visible above the local background. Right, example plot of coctactin and clathrin fluorescence intensity over time during the formation of a single clathrin-coated pit in the cell shown at left (Video S5). (B and C) Examples of cortactin recruitment during Di-T (B) and VSV (C) uptake. BSC1 cells transiently expressing mCherry-LCa (red) and cortactin-EGFP (green) were inoculated with Alexa Fluor 647-labeled Di-T or VSV, and images were acquired as in A. Left, split-channel kymograph views of protein and virion (blue) fluorescence intensity over time (Wideos S6, S7). Images in the right-hand panels show a snapshot of the maximal cortactin on clathrin fluorescence, and white arrowheads highlight the peak cortactin signal. Right, plots of the cortactin and clathrin fluorescence intensity over time for each internalization event. (D) Relative peak fluorescence intensity of cortactin in cells co-expressing mCherry-LCa and cortactin-EGFP. At its best-transfection, samples were separately inoculated with Di-T or VSV particles, and images were acquired as in A. For each cell that was imaged, the maximum cortactin fluorescence associated with \\\\(-\\\\)50 pits lacking virus particles (pits, black) and all pits that internalized a DI-T (left, blue, 3 cells) or VSV (right, red, 5 cells) particle was measured. The data are plotted as described in the legend of Figure 3C, and the number of events is shown above each plot. Numerical values and statistical analyses are provided in Table 1.**\\n\\n'", "CAPTION FIG6.png": "'Figure 6: **Actin polymerization is not required for DI-T internalization.** (_k_) The endocytic fate of virus particles after inhibition of actin polymerization. BSC1 cells stably expressing \u03b12-EGFP (green) were treated with 6.3 \u03bcM IntB for 12 min. and inoculated with DI-T and VSV particles in the continued presence of IntB. Time-lapse images of a single cell were acquired at 4 s intervals for 692 s, and an 8.8 \u00d75.0 \u03bcM2 area of the cell surface is shown. The upper panels show the complete internalization of a DI-T particle (blue, blue arrowheads), where +0 s indicates the first frame of the time-lapse series. The lower panels show the subsequent capture but failed internalization of 2 VSV (red, red arrowheads) particles on the same area of cell membrane (time scale continued from above) (Video S8). (_k_) AP-2 fluorescence intensity for the events shown in A. Note that the adaptor fluorescence associated with the DI-T particle (blue) and a conventional coated pit (black) that formed within the same membrane area peak and disappear normally, while the adaptor signal associated with the upper-most VSV particle (red) does not, signifying failed internalization. (_C_) Effect of latB on the efficiency of virus capture and internalization. BSC1 cells stably expressing \u03b12-EGFP were treated and imaged as described in A. Left, the percentage of virus particles that were captured by a clathrin structure after attachment. Right, the percentage of captured virus particles that were successfully internalized within 300 s of capture (see D. for details). Cumulative data are from 5 cells. (_D_) Effect of latB on the lifetime and peak fluorescence intensity of AP-2 in clathrin structures. Data were acquired as described in A. and displayed as in the legend of Figure 3C. Left, relative lifetime of AP-2 in structures that lack (pits, black) or capture a virus particle. Inset shows a rescaled distribution of the pit and DI-T internalization events. Right, maximum fluorescence intensity of AP-2 in the events at left. Data are from 4 of the 5 cells analyzed in C, as thermal drift during imaging prevented accurate fluorescence intensity measurements in one cell. The number of events in each category is shown above the corresponding plots at right. DI-T (blue) data consists only of productive internalization events. VSV events are categorized as productive internalizations (VSV entry, red) or non-productive captures (trapped VSV, red). A non-productive capture is defined as a stable colocalization between a spot of AP-2 and a VSV particle that began at least 300 s before the last captured image and did not result in virus uptake before cessation of imaging. The 300 s cutoff was chosen because a majority (22/24) of productive internalizations occurred within 300 s of capture. Captures in which the final image frame was acquired before 300 s elapsed were excluded from the analysis, as the eventual endocytic fate of the particle cannot be predicted.\\n\\n'", "CAPTION TAB1-1.png": "'\\n\\n**Table 1.** Summary of kinetic and fluorescence intensity data'", "CAPTION TAB2-1.png": "\"Kinetic and fluorescence values are provided as the main +/- SD for all events in a given context. The number of experiments and cells analyzed is indicated. The absolute lifetime are expressed in seconds (d). The % lifetime and % maximum fluorescence intensity values were calculated as described in the legend of Figure 3C. The % maximum fluorescence intensity of correlation is provided for events analyzed in cells co-expressing mCherry-1Ca and correlation-6FP. A two-tailed Student's +-test was used to determine whether data from 2 categories differ in a statistically significantly manner.\\n\\n(f) denotes a statistical difference with a p-value <0.005 and >0.00005 when comparing data in a given category to data for pick lacking virus in the same category. (g) similarly denotes a p-value <0.00005. All values for _V_SD are significantly different (p<0.005 for % max AP-2 fluorescence; p<0.00005 for all other categories) from those measured for DT-T particles in the same context. ND, not determined.\\n\\ndoi:10.1371/journal.ppat.1001127.1001\"", "CAPTION FIG7.png": "'\\n\\n**Figure 7.** **Model of DI-T and VSV entry.** DI-T (above] and VSV (below) particles engage host cells through interactions between the viral surface glycoproteins and unknown cellular receptor moieties. Following attachment, both particle types undergo slow diffusion diffusion coefficient \\\\(-5\\\\times 10^{-11}\\\\) cm\\\\({}^{2}\\\\)\\\\(\\\\varepsilon^{-1}\\\\); an the cell surface for an average of \\\\(-2\\\\) min before being captured by a clathrin-coated pit. For DI-T, continued clathrin assembly drives complete particle envelopment by the plasma membrane and leads to virus endocytosis. In contrast, the presence of a VSV particle in a coated pit physically prevents complete membrane constriction by the clathrin machinery and causes clathrin assembly to halt prematurely. The force provided by actin polymerization then further remodels the plasma membrane and thereby encloses the virus particle in a partially clathrin-coated vesicle. doi:10.1371/journal.ppa.1001127.g007'", "CAPTION FIG2-1.png": "'\\n\\n**Figure 2.** **Clathrin structures capture VSV and Di-T particles with similar kinetics.** (A) Schematic of clathrin-dependent virus internalization. It. A particle (blue) attaches to receptor moieties: (orange) on the cell surface (black horizontal line), and the virus-receptor complex diffuses in the plane of the membrane. 2. The virus particle is captured by the clathrin endocytic machinery (AP-2, green).\\n\\n'", "CAPTION FIG2-2.png": "\"clathrin, red) after diffusion into an existing clathrin structure (e.g. Genague virus) or entrapment within a clathrin structure that initiates in the close proximity to the virion (e.g. VSW and influenza A virus). 3. Clathrin assembly completes, and the virus-containing pit is screwed from the cell surface in a dynamic-dependent process. Internalization of VSW also requires local actin assembly. Clathrin is rapidly removed from the nascent vesicle, and the vesicle is actively transported further into cell. (B) Example of a complete DI-T internalization event. A single DI-T particle (red) attaches to a BSC1 cell expressing c2-eGFP (green) and diffuses on the cell surface. A dim spot of AP-2 appears beneath the virion, signifying capture of the particle. The AP-2 fluorescence intensity increases as the clathrin coat assembles, and the virus disappears into the cell shortly after the AP-2 signal reaches a maximum (Video S1). Munbered stages correspond to the events described in \\\\(A\\\\). The path of particle motion is depicted as a linear, color-coded trace that progresses with time from blue to red. (C) VSW and DI-T particle capture by clathrin structures in the same cell. BSC1 cells stably expressing c2-eGFP (green) were inoculated with Alexa Fluor 647-labeled DI-T (blue, blue arrowheads) and Alexa Fluor 568-labeled VSW (red, red arrowheads). Time-lapse images were acquired at 4 s intervals using a spinning disk confocal microscope. Left, snapshot of a cell depicting coated pits lacking (white arrowheads) or containing (blue/red arrowheads) virus particles. Right, expanded split-channel views of the region within the dashed box at left. (D) Kinetics of virus capture. BSC1 cells stably expressing c2-eGFP (_T_ cells) or eGFP-LCa (12 cells) were inoculated with VSW and DI-T particles. Images were acquired at 3-4 s intervals as in C, and the time interval between virus attachment and detection of AP-2 or LCa boresath a virion was quantified for productive internalization events. The distribution of capture times is shown for DI-T (blue) and VSW (red) particles, and the mean time to capture \\\\(\\\\langle + \\\\)/\\\\(-\\\\) SD/ for each particle population is provided at right. The kinetics of VSW and DI-T capture are not significantly different (Student's t-test p value = 0.3).\\n\\n\"", "CAPTION FIG1.png": "\"Figure 1.: **Biological properties of DI-T particles.** (A) Structure of VSV and DI-T genomic RNAs. The single-stranded negative-sense RNA genomes are shown in a 3'-5\u2032 orientation. The five viral genes are colored as follows: red nucleocapsid (M) protein gene; orange, phosphoprotein (P) gene; yellow, matrix (M) protein gene; green, glycoprotein (G) gene; blue, large (L) polymerase protein gene. The noncoding genomic terminal leader (Le) and trailer (Tr) regions, which serve as promoters for RNA synthesis and genomic RNA encapsidation, are shown in gray. The DI-T genome comprises 2,208 nts. The 5\u2032 terminal 2,163 nts derive from the 5\u2032 terminus of the parental VSV genome (11,161 nts), and the 3\u2032terminus contains a 45 nts inverted complement of the wild-type Tr (TrC). (B) Electron micrographs of purified VSV and DI-T particles negatively stained with PTA. Middle panel, inset shows an expanded view of virions from the boxed region to facilitate visualization of the viral glycoprotein spikes. Inset scale bar, 100 nm. (C) Vision geometry. The dimensions of individual DI-T (blue) and VSV (red) particles measured from electron micrographs of negatively stained particles. Each open circle represents the measurement for a single particle. Horizontal red lines denote the mean value of each population, and the numerical means (+/- SD) are provided above each plot. (D) Protein composition of purified VSV and DI-T particles. Viral proteins (L, G, P, N, and M) were separated by SDS-PAGE and visualized with Coomassie blue staining. The ratio of N protein to G protein was quantified using ImageJ and is displayed below the gel as a comparative measure of average virion surface glycoprotein density in each particle population. (E) Fluorescence intensity of virus particles labeled with Alexa Fluor dye molecules. Purified DI-T or VSV particles were labeled with Alexa Fluor 647 or 568 and imaged on separate glass coverslips using a spinning disk confocal microscope. The fluorescence intensity of individual spots in a single field of view was quantified, and the distribution of intensity values (in arbitrary units, a.u.) for DI-T (blue) and VSV (red) particles is shown.\\n\\n\"", "CAPTION FIG3.png": "'Figure 3: **Cells internalize Di-T particles using conventional clathrin-coated vesicles.** (A) internalization of VSV and Di-T by the same cell. BSC1 cells stably expressing \\\\(\\\\sigma\\\\)2-eGFP (green) were inoculated with VSV (red) and Di-T (blue) particles, and confocal images were captured at 4 s intervals. The images show the sequential internalization of single VSV (red, red arrowheads) and Di-T (blue, blue arrowheads) particles, followed by the formation of a canonical coated vesicle (white arrowhead) within a 3.5\\\\(\\\\times\\\\)3.5 \u03bcm\\\\({}^{2}\\\\) area of the plasma membrane (Video S2). The first acquired frame of the time-lapse series is designated +0 s, and the capture time of the subsequent images is shown. (B) AP-2 recruitment during the uptake events shown in A. Left, image quadrants depicting AP-2 accumulation over time (left panels) and at the time of maximum AP-2 signal during each event (right panels). Upper panels in each quadrant show the AP-2 channel alone, and the lower panels show overlays of the virus and AP-2 channels. The highlighted pit from A. is indicated with a white arrowhead, and virus particles are colored as in A. Right, fluorescence intensity (in arbitrary units, a.u.t) of AP-2 over time for the events shown at left and in A. The time of AP-2 detection above the local background was set to +-0 s for each event. (C) Kinetics and AP-2 content of endocytic structures. The plots show the relative lifetime (left) and maximum fluorescence intensity (right) of AP-2 during the uptake of coated pits lacking virus (pits, black) or structures that internalized Di-T (blue) or VSV (red) particles (from 4 cells). Values are expressed as percentages to facilitate comparison of viral and nonviral uptake events across multiple cells. Approximately 50 pits lacking virus were analyzed in each cell, and the mean of the measured values was calculated for each parameter. The values for each nonviral and viral uptake event were divided by the mean for pits lacking virus in the same cell, and the resulting values were multiplied by 100. Each open circle represents a single uptake event, and horizontal red lines demark the mean of the compiled population. The number of events is provided above each plot. Numerical values and statistical analyses are provided in Table 1. (D) Clathrin recruitment during virus entry. Left, kymograph views of internalization events from a single BSC1 cell transiently expressing GFP-LCa (Videos 53, 54). Images were captured as in A. and displayed as described in R. Right, fluorescence intensity of eGFP-LCa over time for the events shown at left. (E) Kinetics and clathrin content of endocytic structures. The plots show the relative lifetime (left) and maximum fluorescence intensity (right) of clathrin during the uptake of coated pits lacking virus (pits, black) or structures that internalized Di-T (blue) or VSV (red) particles (from 3 cells). Data were calculated and plotted as described in C. Numerical values and statistical analyses are provided in Table 1.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Figure 4. Electron microscopic images of DI-T and VSV particles in clathrin-coated pits.** (A) Electron micrographs depicting DI-T particles at early (left) or late (right) stages of clathrin-dependent endocytosis. BSC1 cells were incubated with \\\\(-1000\\\\) particles per cell for 10 min. at 37\\\\({}^{\\\\circ}\\\\)C. Cells were then fixed, and samples were processed for ultra thin sectioning and viewed as described in the materials and methods. (B) Electron micrographs of VSV particles at sequential (left to right, top to bottom) stages of clathrin-dependent endocytosis. VSV cells were inoculated with VSV at an _W_OI of \\\\(\\\\xi\\\\), and samples were prepared for analysis at 6 h post-infection.\\n\\ndoi:10.1371/journal.pga.1001127.g004.\\n\\n'", "CAPTION TAB1.png": "'\\n\\n\\\\begin{table}\\n\\\\begin{tabular}{l l} \\\\hline \\\\hline \\\\multicolumn{1}{c}{**Table 1.** Summary of kinetic and fluorescence intensity data.} \\\\\\\\ \\\\hline \\\\hline \\\\end{tabular}\\n\\\\end{table}\\nTable 1: Summary of kinetic and fluorescence intensity data.\\n\\n'", "CAPTION TAB2.png": "'* [18] B.\\n\\n'", "CAPTION FIG2.png": "'Figure 2.: **Cistarius structures capture VSV and D-T particles with similar kinetics.** (U) Schematic of clarity-dependent virus internalization. 1. A particle label attaches to receptor moieties (orange) on the cell surface block horizontal line, and the virus-receptor complex diffuse in the plane of the membrane. 2. The virus particle is captured by the clathrin endocytic machinery (AP-2, green, clathrin, red) after diffusion into an existing clathrin structure to-go. Danger twist of entrapment within a clathrin structure that maintains a force primarily to the vitan tip, 95% and membrane A. 3. Glutrich assembly completes, and the virus-containing tip is screened from the cell surface in a dynamic dependent process. Intercalation of VSV also requires local actin assembly. Glutrich is rapidly removed from the nascent vesicle, and the vesicle is actively perturbed further into cells. (II) Example of a complete D-T internalization event. A single D-T particle (and attach) is a 18-cell experiment 20-24-(95%) (green) and 60% on the cell surface. A final spot of 40-24\\n\\n'"} |
