Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

Abstract

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP₂. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

Fig. 1.. Dynamics of VASP clusters assembled from preexisting IRSp53 clusters on the plasma membrane in filopodium initiation.

(A) Wide-field fluorescence image of a representative Rat2 cell transfected with IRSp53-eGFP and RFP-VASP. Brackets indicate some filopodia where IRSp53 is present along them. White arrows indicate the same filopodia to demonstrate that VASP is enriched in their tips. Scale bar, 5 μm. (B) Time-lapse images of a filopodium formation. Images are magnifications of the indicated area (cyan boxes) in (A). The white arrow indicates the appearance of an IRSp53 cluster followed by a VASP cluster indicated by a cyan arrow at the onset of filopodium formation. White boxes indicate the selected area used to generate outlines of plasma membrane positions over time shown in (C). Scale bar, 2 μm. (C) Colored outlines of membrane positions in the region indicated by the white boxes shown in (B). Total of 27 frames and frame interval of 2 s. (D) Adaptive kymograph maps replot the detected membrane profiles in (C) in the y axis and the corresponding time points in the x axis to show the dynamics of IRSp53 (left) and VASP (right) on the plasma membrane over time. Y axis shows the membrane positions of the proteins, and the x axis shows the time (in seconds; total of 27 frames). Scale bars, 1 μm (y axis). Color maps: Low fluorescence intensity is in blue, and high fluorescence intensity is in red. Circled numbers correspond to the frames indicated in (B).

Fig. 2.. IRSp53 self-assembles into clusters and recruits VASP on PIP₂ membranes.

(A and B) Representative confocal images of GUVs incubated with AX488-labeled IRSp53 (16 nM). GUVs contain brain total lipid extract (TBX) with 0.5% TR-ceramide and either 5% PIP₂ (PIP₂-GUVs) in (A) or 25% 1,2-dioleoyl-sn-glycero-3-phospho-L-Serine (DOPS) (PS-GUVs) in (B). TR-ceramide in magenta and IRSp53 in cyan. Arrows indicate IRSp53 clusters on GUV membranes. Arrowhead indicates an inward membrane tube generated by IRSp53. Scale bar, 5 mm. (C) Sizes of IRSp53 clusters on PIP₂-GUVs and on PS-GUVs. Each data point represents one cluster. PIP₂-GUV: total of 225 clusters; N = 42 GUVs, three sample preparations. PS-GUV: total of 55 clusters; N = 12 GUVs, two sample preparations. Statistical analysis: two-tailed Mann-Whitney test, P = 0.1125. (D) Top: Representative snapshots of CG simulations from PS-like (0% PIP₂), 2% PIP₂-like, and 5% PIP₂-like membranes. Membrane CG beads are in gray, PIP₂-like CG beads are in blue, and I-BAR domains are in red. Scale bar, 50 nm. Bottom: Enlarged areas as indicated by the white dashed boxes. Only the central portion of the I-BAR domain is shown (yellow) to visualize PIP₂ clusters (blue). (E) Probability of I-BAR domain aggregate size to be <5 or ≥5 molecules for membranes shown in (D). (F) Representative confocal images of GUVs incubated with AX488-labeled VASP (yellow) together with (top) or without (bottom) IRSp53 (unlabeled). Protein concentrations: 16 nM IRSp53 and 4 nM VASP. GUVs contain TBX with 0.5% TR-ceramide (magenta) and 5% PIP₂. Heatmaps in (A) and (B) for IRSp53 signals and (F) for VASP signals; low fluorescence intensity is in blue, and high fluorescence intensity is in red. Scale bars, 5 μm.

Fig. 3.. IRSp53 and VASP synergistically drive the formation of actin-filled membrane protrusions.

(A to D) Representative GUVs (membranes, magenta; actin, green) incubated with (A) all proteins, (B) where actin was labeled instead with AX488 phalloidin, and where (C) IRSp53 and (D) VASP were excluded. Cartoon in (A) depicts GUVs incubated with all proteins (CP and profilin not shown). All proteins: IRSp53 (16 nM), VASP (4 nM), actin (0.5 μM; 10 to 27% AX488 labeled), fascin (250 nM), CP (25 nM), and profilin (0.6 μM). GUV composition: TBX, 0.5% TR-ceramide, and 5% PIP₂. Scale bars, 5 μm. (D) Right: Pixel-averaged actin signals on GUVs. “No VASP,” N = 29 GUVs; “With VASP,” N = 29, one preparation (see fig. S12A for another two preparations). (E) Percentages of GUVs having actin-filled tubes with (“With VASP”) and without (“No VASP”) VASP. “With VASP,” N = 56, 39, and 45; “No VASP,” N = 42, 41, and 33; three preparations. Statistical analyses: chi-square test on pooled data, P < 0.0001; paired t test, P = 0.0585. (F and G) Percentages of tube-positive GUVs in the absence (“No actin”) and presence (“With actin”) of actin. “With actin” GUVs were counted only when having actin-filled tubes (*). (F) “No phalloidin” corresponds to AX488-labeled actin. “No actin,” N = 39 and 60; “With actin,” N = 45 and 56; two preparations. Statistical analyses: chi-square test on pooled data, P < 0.0001; paired t test, P = 0.3072. (G) In the presence of AX488 phalloidin and no AX488-labeled actin. “No actin,” N = 31, 31, 26, and 39; “With actin,” N = 54, 57, 41, and 56; four preparations. Statistical analyses: chi-square test on pooled data, P < 0.0001; paired t test, P = 0.0054.

Fig. 4.. Fascin facilitates filopodium growth and prevents filopodium retraction.

(A) Left: Representative images of GUVs incubated with all protein ingredients (IRSp53, VASP, actin, CP, and profilin) besides fascin. Right: Percentages of GUVs having actin-filled tubes in the absence (“No fascin”) and presence (“With fascin”) of fascin. GUV composition: TBX, 0.5% TR-ceramide, and 5% PIP₂. “No fascin,” N = 53, 32, and 54 GUVs; “With fascin,” N = 56, 45, and 39; three sample preparations. Statistical analyses: chi-square test on pooled data, P = 0.8652; paired t test, P = 0.6906. (B) Wide-field fluorescence image of a Rat2 cell transfected with IRSp53-eGFP and mCherry-fascin. (C) Time-lapse images of the formation of a filopodium. Images are magnifications of the indicated area (white dashed boxes) in (B). White arrows indicate the appearance of IRSp53 clusters followed by fascin recruitment. Time is in seconds. (D) Left: Representative kymograph of IRSp53 and fascin fluorescence signals in a growing filopodium showing the growth before (number 1) and after (number 2) the presence of fascin. Right: Quantification of filopodial growth rate before (“Before fascin arrived”) and after (“After fascin arrived”) fascin recruitment. N = 26 filopodia. Statistical analysis: Mann-Whitney nonparametric test, P = 0.00001633. (E) Frequency of filopodium retractions in IRSp53-eGFP–expressing Rat2 cells transfected with either an empty mCherry plasmid (“Control”) or mCherry-fascin (“Fascin”). The frequency of each event was calculated for the period of filopodium growth as the number of retractions per second. “Control,” N = 13 filopodia; “Fascin,” N = 30. Statistical analysis: Mann-Whitney nonparametric test, P = 0.000083802. Scale bars, 5 μm (A) and 2 μm (B and C).

Fig. 5.. IRSp53’s I-BAR domain is robustly recruited into pulled membrane nanotubes.

(A) Experimental setup for pulling membrane nanotubes using a concanavalin A (ConA)–coated bead trapped in an optical tweezer (OT). Rat2 fibroblasts, expressing eGFP fusions (green) of either IRSp53’s I-BAR domain or the full-length (FL) IRSp53 protein, were labeled with CellMask Deep Red plasma membrane stain (magenta); protein enrichment in the membrane nanotube was monitored by confocal fluorescence microscopy using single-photon avalanche detectors (cts, counts). (B) Representative confocal image of a pulled membrane nanotube from a Rat2 cell expressing IRSp53’s I-BAR domain showing high enrichment of the I-BAR domain. (C) Top: Calculated sorting map of the nanotube in (B) with low sorting (S) values in blue and high S values in red. Bottom: Plot of the maximum sorting value at each pixel position along the length of the nanotube (white bracket in the sorting map) and the mean sorting value for the protein (S_avg). (D) Measured S_avg values for IRSp53’s I-BAR domain in pulled nanotubes (N = 19 nanotubes). S_avg > 1 (dashed black line) indicates protein enrichment. Black solid line, mean of the data points. Dashed white circles in the figure outline the trapped bead. Scale bars, 5 μm.

Fig. 6.. IRSp53 is recruited into membrane nanotubes pulled from highly active cellular zones and coincides with actin filament assembly.

(A and B) Representative confocal images of pulled membrane nanotubes from Rat2 cells expressing the full-length IRSp53 protein (IRSp53-eGFP). (A) Pulled nanotubes near cellular zones of active membrane remodeling, such as membrane ruffling (white arrowheads), exhibit recruitment of IRSp53 in the nanotube. (B) Pulled nanotubes near non-active zones instead show no recruitment of IRSp53. (C) Mean sorting values for a given nanotube, S_avg, were determined from active and non-active zones for IRSp53-expressing cells. Dashed black line, S_avg = 1. Black solid lines, mean of the data points. Active zone, N = 13 nanotubes; non-active zone, N = 20 nanotubes. (D) IRSp53 enrichment corresponds with eventual actin development within the pulled nanotube. Representative sorting (S) map (left) and the corresponding force plot (center) for a nanotube showing high IRSp53 sorting. Peaks in the force plot (black arrowheads) are signatures of actin in the tube and arise when retrograde flows outcompete actin polymerization (at the nanotube tip), causing bead displacement toward the cell body and hence a rise in the force (right; i and ii). (E) Distribution of the force peak magnitudes (ΔF). Sample size, 100 peaks. (F) Representative sorting map (left) and the corresponding force plot (right) for a nanotube showing no IRSp53 enrichment and hence no actin development. Color maps: Low S values are in blue, and high S values are in red. Dashed white circles in the figure outline the trapped bead. Scale bars, 5 μm.