Fig 1: CYRI proteins localize to macropinocytic structures prior to RAB5 arrival. (See Video 2.) (A and B) Representative images of live COS-7 cells expressing P17-GFP-CYRI-B (scale bar = 10 µm). Tubular and vesicular structures are highlighted in zoomed panels, and quantification of their sizes is shown in B (vesicles, n = 16 events in 3 cells; tubules, n = 22 events in 4 cells; scale bar = 5 µm). (C–E) Time sequence of live HEK293T cells (scale bar = 10 µm) coexpressing P16-GFP-CYRI-A (cyan) and mCherry-RAB5A WT (magenta). Arrowhead points to vesicular structures (C; scale bar = 5 µm). The dynamics of each protein is reported by its normalized intensity plot (D) and lifetime (n = 84 events in 7 cells; E). (F and G) COS-7 cells (scale bar = 10 µm) coexpressing P17-GFP-CYRI-B (cyan) and mCherry-RAB5A WT (magenta). Time sequence corresponding to the white dotted square area is shown in the bottom panel (scale bar = 5 µm). Arrowhead points to tubular and vesicular structures, and intensity profile along the yellow line is plotted in G. (H–M) Time sequence images HEK293T (H) and CHL-1 cells (K) expressing P16-GFP-CYRI-A and incubated with dextran 70 kD (scale bar = 10 µm). Yellow arrowheads indicate macropinocytic events positive for both CYRI-A and dextran signals (scale bar = 5 µm). Quantification showing the majority of CYRI-A–positive vesicles are also dextran-positive in HEK293T (I; 88%, n = 6 cells) and CHL-1 (L; 100%, n = 6 cells) and their sizes (J and M; n = 53 events in 6 cells in HEK293T; n = 57 events in 6 cells in CHL-1). Red line indicates the average value. (N and O) HEK293T cells expressing either GFP control or P16-GFP-CYRI-A and incubated with dextran 70 kD. The size of dextran-positive vesicles in GFP (n = 163 events in 4 cells) is the same as that of CYRI-A–positive vesicles (n = 75 events in 7 cells). Events from each cell are color-coded. Unpaired t test. Mean ± SD.
Fig 2: CYRI-A and CYRI-B cooperatively regulate cell shape and migration.(A) Immunofluorescence images of control (pLKO), single KO, and DBKO of CYRIs in A-673 cells. Cells stained for F-actin with phalloidin. Orange arrowheads indicate C-shape cells. Scale bar = 50 µm. (B) Western blots showing the efficiency of single KO and DBKO of CYRIs. GAPDH as loading control. (C) Quantification of cell shape (left) and spread area (right) of A from at least 50 cells per experiment from three independent experiments. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; **, P < 0.01; ****, P < 0.0001. (D and E) Migration analysis of CYRI CRISPR cells on a 2D fibronectin substrate (random migration) or in 3D CDM. Data from at least 30 cells per experiment from three independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ****, P < 0.0001. (F) Reexpression of CYRI-A in DBKO A-673 cells reduces their speed to the original values but does not affect the control pLKO cells. Data from at least 30 cells per experiment from three independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; ****, P < 0.0001. (G and H) Wound healing assay comparing the control pLKO, single KO, and DBKO CYRI cells. Data from at least four independent experiments. Each experiment is color-coded. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison test. ns, P > 0.05; ***, P < 0.001; ****, P < 0.0001. Orange dotted lines highlight the edge of the cell monolayer. (I) Proliferation assay using the Incucyte system of control pLKO, single KO, and DBKO CYRIs in A-673 cells. Data from at least three independent experiments. Mean ± SEM.
Fig 3: CYRI-A-colocalizes with plasma membrane-associated nascent macropinocytic structures. (See Video 3.) (A–D) Time sequence images of COS-7 cells expressing P16-GFP-CYRI-A or P17-GFP-CYRI-B (cyan) and either mCherry-tagged CLC15 (clathrin light chain 15; A and B), Caveolin-1 (C), or ARF1 (D). Scale bar = 10 µm for full-size image and 5 µm for zooms. (E and F) Time sequence images of live COS-7 cells coexpressing either P16-mCherry-CYRI-A WT or P16-mCherry-CYRI-A RRDD mutant (magenta) and P16-GFP-CYRI-A WT (cyan). (G–L) COS-7 cells coexpressing P16-GFP-CYRI-A (cyan) and two independent PIP3 reporters (magenta), PH-Grp1 (G–I) or PH-Btk (J–L; n = 31 events in 3 cells for Grp1; n = 9 events in 1 cell for Btk). Red line represents the average value. Scale bar = 10 µm for full-size image and 5 µm for zooms. (M and N) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mScarlet-Lck (labeling the plasma membrane; magenta). The time Lck resides on the vesicles before CYRI-A is recruited is quantified in N (n = 48 events in 10 cells). Scale bar = 10 µm for full-size image and 3 µm for zooms. Red line indicates the average value.
Fig 4: CYRI-A regulates actin dynamics at macropinocytic structures. (See Video 4, Video 5, and Video 6.) (A) Still images of COS-7 cells coexpressing either GFP (negative control) or P16-GFP-CYRI-A (cyan) and LifeAct-RFP (magenta). Scale bar = 10 µm for full-sized image and 5 µm for zooms. (B) Time sequence images showing the dynamics of P16-GFP-CYRI-A and actin at the macropinocytic structure in COS-7 cells. Graph shows normalized signal intensities over time. Black arrows denote peak actin and CYRI-A signals. Scale bar = 5 µm. (C) Normalized signal intensities over time between P16-GFP-CYRI-A and LifeAct signal in HEK293T cells. (D) Lifetime of actin before and after P16-GFP-CYRI-A is recruited in HEK293T cells (before CYRI-A, n = 25 events in 10 cells; with CYRI-A, n = 34 events in 10 cells). Red lines indicate the average value. (E–G) Lifetime of actin on macropinocytic structures ± expression of P16-GFP-CYRI-A in CYRI DBKD COS-7 cells. Scale bar = 1 µm. Number of actin-positive structures in cells ± P16-GFP-CYRI-A expression (n = 9 cells; F). Lifetime of the actin signal on macropinocytic structures ± P16-GFP-CYRI-A signal (actin alone, n = 43 events in 9 cells; actin with CYRI-A, n = 33 events in 8 cells). (H) Macropinocytosis assay in siRNA-treated COS-7 cells. Scr, scramble. Black dots are internalized dextran. Black dashed lines indicate the boundary of the cell clusters. Scale bar = 30 µm. (I) Macropinocytic index of H. Data are from at least 10 different fields of view per experiment from a total of three independent experiments (color-coded by experiment). Two-tailed unpaired t test. Mean ± SD. (J and K) Expression of P16-mCherry-CYRI-A in control COS-7 cells, DBKD COS-7 cells, and P16-mCherry-CYRI-A RRDD mutant (non-RAC1 binding mutant) cells showing dextran 70-kD uptake capacity of the cells. Data are from =10 different fields of view for a total of three independent experiments. Each experiment is color-coded. Mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparison test. ns, P > 0.05. (L and M) Lifetime of P16-GFP-CYRI-A on macropinosomes ± 1 µM of Latrunculin A (LatA) or Cytochalasin D (CytoD) in COS-7 and HEK293T cells. At least five cells per experiment from three independent experiments (color-coded). Mean ± SD. Mann–Whitney U test.
Fig 5: CYRI-A suppresses cell spreading and leading-edge Arp2/3 complex recruitment.(A) Immunofluorescence images of COS-7 pLKO (control) and CYRI-B CRISPR KO lines (ex3 or ex4.1) expressing either GFP-FLAG or CYRI-A-FLAG vector and stained for Arp2/3 complex (anti-ArpC2, yellow), FLAG-tag (magenta), and F-actin (phalloidin, cyan; scale bar = 20 µm). Dotted square denotes zooms (scale bar = 5 µm). (B and C) Cell spread area and Arp2/3 signal at the leading edge in COS-7 cells expressing either GFP-FLAG (squares) or CYRI-A-FLAG (circles). Data from at least 10 cells per experiment and three independent experiments, each colored separately in blue, orange, and green. Statistical analyses using two-tailed unpaired t test. Mean ± SD. (D) Representative Western blot showing relative expression of GFP-FLAG and CYRI-A-FLAG in COS-7 detected with anti-FLAG antibody. GAPDH as loading control.
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