{"CAPTION FIG2.png": "'\\n\\n**Fig. 2. Dynamic assembly of GRAF1 at the leading edge.** (A) Immunobists detecting endogenous and recombinant GRAF1 in Flp-fn T-REx HeLa GFP-GRAF1, GRAF1 and GRAF1-GFP cell lysates after induction with the eluted doxycycline (Deoxy) concentrations for 24 h. A deoxycycline concentration of 1 ng/ml was chosen for all subsequent experiments. (B) Fluorescence micrograph detecting GRAF1-GFP, corresponding to the last frame of a live-cell spinning disc, confocal microscopy acquisition. As the leading edge, tracks of structure movement and duration over three are illustrated as colour-coded lines. Raprecardation frames from the same acquisition are presented as a time series. Scale bar: 10 um. (C) Duration time of GRAF1-GFP structure tracks derived from acquisitions exemplified in B. 19 cells from three independent experiments were analyzed. The bar graph depicts the distribution of the means-d values derived from each evaluated cell (D) Duration time of GRAF1-GFP and GFP-GRAF1 structure tracks derived from live-cell TIRF microscopy acquisitions. Bar graphs depicts the distribution of the mean+s.d. values derived from each cell included in the respective analysis. Three independent experiments were analyzed (Mann-Whitney U terz, r=11-12 cells, a=0.08, ris, not significant). (E) Fluorescent micrograph showing GRAF1-GFP and mCherry-tagged clathrin light chain (CLC) at a protrusion, corresponding to core frame from a live-cell TIRF microscopy acquisition. Traces visualise the detected structure movement in the respective channel. The edge of the cell is outlined in the upper panels. ArroMethods highlight GRAF1 assemblies. Scale bar: 2 um.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n**Fig. 3.**Inhibition of **G**dc42 function and **ac**ln polymerization effects **G**RAF1 carrier processing. (A,B) Relative normalization of aminodextrin-S-G-C/V**400 and transferrin-S-S-C/V**400 after 30 min uptake in Fip-ln T-R**2x HeLa GFP-G**RAF1 cells depleted at AP-2, Cdc42 or G**RAF1 by siRNA transfection (A), as confirmed by the immunodetection of the respective protein in camparan to clathrin heavy chain (CHC) in the cell lysates (B). The cargo uptake was normalized to the corresponding control _a_-RNA-transfected sample and the significance in relation to this sample was determined by analyses of at least three independent experiments (means,d; Mann-Whitney U tests, _n_=3-4 samples, a=0.0%, ns, not significant; \"pe0.0%). (C) Fluorescent micrographs depicting G**RAF1-GFP** in control cells and cells depleted of Cdc42. The ratio of cells with G**RAF1** in at least one tubular structure (i.e. a structure with a length >2 mm) was quantified on the basis of three independent experiments (means,d - Chi-square test, _n_>480 cells, a=0.0%, +++p<0.0001). (D) Ratio of cells with GFP-G**RAF1** in tubular structures after treatment with DMSO (Vehice) or withostatin (Vhisk), for the indicated time interval. Cells from three independent experiments were included in the analysis (means,d.; Chi-square tests, a=0.0%, +++p<0.0001). (E) Cordocal stack at a GFP-G**RAF1-expressing cell after 20 min dissociation treatment, with presented top and slice views positioned as indicated (white arrow). Scale bar. 10 mm.\\n\\n'", "CAPTION FIG1.png": "'\\n\\n[MISSING_PAGE_POST]\\n\\n'", "CAPTION FIG4.png": "'Figure 4: **GRAF1 recruitment to Cdc42** microdomains coincides with a local decreasing plasma membrane association of the GTPase. \\\\(\\\\langle\\\\Delta\\\\rangle\\\\) Time series from a live-cell spinning disc confocal microscopy acquisition, visualizing a protrusion from a Fip-In T-REx HeLa GRAF1-GFP call co-expressing mCherry-Cdc42. White arrows mark detected GRAF1 assemblies. (B) Intensity profiles of the green and red channels, calculated over time as described in the Materials and Methods, for the first-appearing GRAF1 assembly in A. The grey area highlights the peak duration for the green channel. The insert visualizes the ROI used to derive the plotted fluorescence intensity curves. (C-F) Parameters calculated for single GRAF1 assemblies on the basis of the corresponding intensity peak recorded within the intensity profiles derived from live-cell acceptance, as exemplified in A and B. The three points for the intensity peak start, maximum and end were defined for each structure and channel as described in the Materials and Methods. 34\u201336 GRAF1 structures from three cells were included in the analysis and results are meaning.d.\\n\\n'", "CAPTION FIG7-2.png": "'\\n\\n**Fig. 7. Cdc42 GTPase deficiency disrupts the maturation of internalized GRAF1 carriers.** (A) Fluorescence micrograph representing the first frame of a live-cell confocal acquisition detecting dextran-Alexa-Flucr-555 and GFP\\\\({}_{\\\\infty}\\\\) = GRAF1 in Fip-In T-REx HaLa cells co-expressing Myc-Cdc42-Q61L (upper pannels). The acquisition was started after a 5-min incubation with the cargo followed by medium exchange. Analysis on the basis of four captured cells revealed dextran enrichment in 31\\\\(\\\\pm\\\\)16% (means.e.m.) of the identified GRAF1 structures. Areas 1 and 2 from the acquisition are presented as time series (lower panels). Red stars indicate cells affected by Myc-Cdc42-Q61L Scale bar: 10 mm. (B) Epifluorescence micrograph of GFP-GRAF1 and dextran structures after a 5-min uptake in two cells, one defined as phenotypically unaffected (control) and the other defined as affected by Myc-Cdc42-Q61L (red star). Scale bar: 10 mm. (C,D) Quantification of the total dextran uptake and descriptive parameters for dextran-positive compartments for comparison of GFP-GRAF1 cells unaffected and affected by the Cdc42-Q61L transfection, as defined in B, to circumvent inter-sample variation of cargo fluorescence. Error bars represent the s.e.m. from three independent experiments (Mann-Whitney U tests, n=12-14 cells, a=0.05; ns, not significant; \"P<=0.01). (E) Relative internalisation of aminodextrin-S-S-CW800 and transferrin-S-S-CN800 after a 30-min uptake in Fip-In T-REx HaLa GFP-GRAF1-R412D cells. The cargo uptake of the mutant-expressing cells collected from at least three independent experiments was normalised to the corresponding wild-type sample and the significance in relation to this sample was assessed (means.d.; Mann-Whitney U tests, _n_=3-4, a=0.05; ns, not significant). (F) EEA1 intensity per endosome and number of EEA1-positive endosomes per GFP-GRAF1 cell transfected with mCherry-tagged Cdc42 or Cdc42-Q61L. Three independent experiments were analyzed [mean +-s.d.; left diagram, Student\\'s +-test, _n_>13,500 EEA1-stained endosomes (from 50 cells), a=0.05; ns, not significant; right diagram, Mann-Whitney U test, _n_=3 cells, a=0.05; ns, not significant). Bars in the left graph depict the distribution of the mean values derived from each experiment. (G) Confocal fluorescence micrographs of GFP-GRAF1 cells transfected with mCherry-Cdc42-Q61L and immunostained with anti-EEA1 antibody. Scale bar: 5 mm. (H) Ratio of dextran-filled Rab6 compartments in BFP-Rab5-transfected GFP-GRAF1 cells without or with co-expressed Myc-Cdc42-Q61L after a 5-min cargo uptake. Cells from three independent experiments were included in the analysis [means.d.; Mann-Whitney U test, _n_>5 captured frames (at least 5 cells), a=0.05; \"P<=0.05). (I) Representative time series from a live-cell spinning disc confocal microscopy acquisition visualising the dynamics of dextran-filled internal compartments in GFP-GRAF1 cells co-expressing BFP-Rab5 and Myc-Cdc42-Q61L. The yellow arrow marks a Rab6 vesicle devoid of dextran, adjacent to a cargo-filled GRAF1 compartment pointed out by the magenta arrow. The turquoise arrow marks a second GRAF1-labeled structure devoid of dextran, which over the depicted time range fuses with the first GRAF1 compartment (specifically note the redistribution of cargo between these two compartments). Scale bar: 2 mm.\\n\\n'", "CAPTION FIG7.png": "\"\\n\\n**Fig. 7.** **Cdc42 GTPase deficiency disrupts the maturation of internalized **GRAF1 carriers**. (A) Fluorescence micrograph representing the first frame of a live-cell confocal acquisition detecting dextran-Alexa-Fluco-565 and GFP-GRAF1 in Fip-In T-REk HeLa cells co-expressing Myc-Cdcd42-Q6 IL (upper panels). The acquisition was started after a 5-min incubation with the cargo followed by medium exchange. Analysis on the basis of four captured cells revealed dextran enrichment in 31+-16% (means.s.m.) of the identified GRAF1 structures. Areas 1 and 2 from the acquisition are presented as time series (lower panels). Red stars indicate cells affected by Myc-Cdcd42-Q6 IL. Scale bar: 10 um. (B) Erythroscence micrograph of GFP-GRAF1 and dextran structures after a 5-min uptake in two cells, one defined as phenotypically unaffected (control) and the other defined as affected by Myc-Cdcd42-Q6IL (red star). Scale bar: 10 um. (C,D) Quantification of the total dextran uptake and descriptive parameters for dextran-positive compartments for comparison of GFP-GRAF1 cells unaffected and affected by the Cdc42-Q6IL transfection, as defined in B, to circumvent inter-sample variation of cargo fluorescence. Error bars represent the s.e.m. from three independent experiments (Mann-Whitney U tests, _n_=12-14 cells, _a_=0.05; ns, not significant; *P<0.01). (E) Relative internalisation of aminod dextran-S-S-CWN800 and transferrin-S-CWN800 after a 30-min uptake in Fip-In T-REk HeLa GFP-GRAF1-R412 cells. The cargo uptake of the mutant-expressing cells collected from at least three independent experiments was normalised to the corresponding wild-type sample and the significance in relation to this sample was assessed (means.s.d.; Mann-Whitney U tests, _n_=3-4, _a_=0.05; ns, not significant). (F) EEA1 intensity per endosome and number of EEA1: positive endosomes per GFP-GRAF1 cell unaffected with mCherry-tagged Cdc42 or Cdc42-Q6IL. Three independent experiments were analysed [means +s.d.; left diagram, Student's +Rest, _n_=13,500 EEA1-stained endosomes (from 60 cells), _a_=0.05; ns, not significant; right diagram, Mann-Whitney U test, _n_=5 cells, _a_=0.05; ns, not significantly. Bars in the left graph depict the distribution of the mean values derived from each experiment. (G) Confocal fluorescence micrographs of GFP-GRAF1 cells transfected with mCherry-Cdcd42-Q6 IL and immunostained with anti-EEA1 antibody. Scale bar: 5 um. (H) Ratio of dextran-filed Rab5 compartments in BFP-Rab5-transfected GFP-GRAF1 cells without or with co-expressed Myc-Cdcd42-Q6IL after a 5-min cargo uptake. Cells from three independent experiments were included in the analysis [means.d.; Mann-Whitney U test, _n_=5 captured frames (at least 5 cells), _c_=0.05; *P<0.05). (I) Representative time series from a live-cell spinning disk confocal microscopy acquisition visualizing the dynamics of dextran-filed internal compartments in GFP-GRAF1 cells co-expressing BFP-Rab5 and Myc-Cdcd42-Q6IL. The yellow arrow marks a Rab5 vesicle devoid of dextran, adjacent to a cargo-filled GRAF1 compartment pointed out by the magenta arrow. The turquoise arrow marks a second GRAF1-labelled structure devoid of dextran, which over the depicted time range fuses with the first GRAF1 compartment (specifically note the redistribution of cargo between these two compartments). Scale bar: 2 um.\\n\\n\"", "CAPTION FIG5.png": "'\\nFig. 5: **Interaction with transition state Cdc42 enforces membrane assembly of GRAF+.** (A) Ratio of Fig-In T-Rea HeLa cells showing a phenotype of abundant (>15) GFP-GRAF+1 or GFP-GRAF-1.R412D structures in the absence (-) or presence of co-expressed Coral/Hue-tagged small GTPases and mutants thereof. For statistical analysis, cells from at least three independent experiments were included. The results were compared to the GRAF1 samples expressing wild-type Cdc42, or in the case of GRAF1-R412D cells, were related to the corresponding GRAF1 samples (means.d.; Chi-square tests, _n_>=0.05, ns, not significant; \"\"PcO.0001). (B) Maximum intensity projections of cordocal stacks detecting GFP-GRAF1 in cells co-expressing the indicated _Coral/Hue-_tagged Cdc42 proteins. (C) Cocomas-stained SDS-PAGE gel from a platform experiment using purified GST GST or a GST-Tagged GRAF1 GAP-domain (GAP) as bait, and purified Cdc42 or Cdc42-Q61L loaded with GTP-S or GDP in the presence of AIF, (GDP/AIF, as grey). The bound (B) and unbound GTPases (J) were detected. (D) Maximum intensity projections of cordocal stacks detecting GFP-GRAF1 or indicated truncates (the BAR-PH-GAP and BAR-PH domains) and _Coral/Hue\u2013_C_dc42-Q61L in transfected HeLa cells. (E) Percentage volume of GFP\u2013GRAF1 proteins colocalized with Coral/Hue\u2013_C_dc42-Q61L, as visualised in D. Three independent experiments were analysed (means.d.; Kruskal-Wallis test, Dunn\u2019s post test, _n_=15 cells, _\u03b1_=0.05; ns, not significant: \"\"PcO.0001). Scale bars: 10 \u03bcm.\\n\\n'", "CAPTION FIG7-1.png": "'\\n\\n**File T:** See text page for text.\\n\\n'", "CAPTION FIG6.png": "\"Figure 6: **Cdc42 GTPase deficiency affects GRAF1 carrier dynamics after their formation**. (A) Fluorescence micrographs depicting the start frames from the-cell TIRF microscopy acquisitions of GFP-_GRAF1 structures in Fb-in T-REx HeLa cells transfected with the indicated _Coral/Hue-tagged Cdc42 constructs (upper panels). The corresponding structure tracks over time, derived from the acquisitions, are represented as colour-coded lines (lower panels). Structures detected over the entire basal cell surface were included in the track analysis. (B\u2013E) Quantification of the number and dynamic parameters of GFP-_GRAF1_ structure tracks from acquisitions exemplified in A. Three independent experiments were analysed [Student's +-tests on log10-transformed data, \\\\(n\\\\)2-136 structure tracks (from four cells), _\u03b1_=0.05; ns, not significant; ***P>0.0001]. Bar graphs in C\u2013E depict the distribution of the means.d, values derived for each cell included in the respective analysis. (F) Fluorescence micrograph showing the first frame from a live-cell spinning disc confocal microscopy acquisition of GFP-_GRAF1_ structures in cells transfected with the indicated _mCherry_-tagged Cdc42-Q61L. Speeds measured over the detected GRAF1 tracks are represented as colour-coded lines. Magnifications of areas 1 and 2 are visualised as a time series to highlight the mobile membrane structures positive for both proteins. The white arrow marks a structure at the cell surface, the yellow arrow follows the lateral movement of an internal structure and the red arrow points out the fission of a tubular structure from the cell surface. (G) Quantification of duration of colocalisation between GFP-_GRAF1_ membrane assemblies and respective indicated _mCherry_-tagged Cdc42 protein, based upon track analyses performed on live-cell spinning disc confocal microscopy acquisitions like that exemplified in F. Cells from two independent experiments were analysed and the means.d. is indicated [Student's +-test on log10-transformed data, \\\\(n\\\\)2-29 structure tracks (from three cells), _\u03b1_=0.05; ***P>0.0001]. Scale bars: 10 \u03bcm.\\n\\n\""}