Myosin-X recruits lamellipodin to filopodia tips

Abstract

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Keywords: Cargo transport; Filopodia; MYO10; Molecular motor; RAPH1.

Fig. 1.

Mass spectrometry analyses identify RAPH1 as a putative MYO10 binder. (A,B) Mass spectrometry (MS) analysis of GFP–MYO10^FERM- and GFP–Talin^FERM-binding proteins. Comparison of the GFP–MYO10^FERM dataset to GFP (A) and GFP–Talin^FERM (B) datasets are displayed as volcano plots where the fold-change enrichment is plotted against the significance of the association (see Table S1 for the MS data). The volcano plots were generated using VolcaNoseR (Goedhart and Luijsterburg, 2020). (C) U2-OS cells transiently expressing GFP–MYO10–BioID were plated on fibronectin (FN) in the presence of biotin for 24 h, fixed, stained for biotinylated proteins (using streptavidin), F-actin, and DAPI, and imaged using a spinning disk confocal (SDC) microscope. Scale bars: 25 µm (main); 5 µm (magnification). Note that only one cell in this field of view expresses the GFP–MYO10–BioID construct. Images are representative of three biological repeats. (D) U2-OS cells stably expressing GFP–MYO10–BioID or GFP–MYO10 were plated on FN for 24 h in the presence of biotin. Cells were then lysed, and biotinylated proteins were purified using streptavidin beads. Recruited proteins were analyzed using western blotting and MS (see Table S1 for the MS data). Western blots are displayed (representative of five biological repeats). (E) Venn diagram highlighting the overlap of MYO10-enriched proteins identified from the indicated MS datasets. (F) GFP pulldown in U2-OS cells expressing GFP–MYO10^FERM, GFP–Talin^FERM or GFP alone. RAPH1 recruitment to the bait proteins was then assessed by western blotting (representative of three biological repeats). (G) U2-OS cells stably expressing GFP–MYO10–BioID or GFP-MYO10 were plated on FN for 24 h in the presence or absence of biotin. Cells were then lysed, and biotinylated protein purified using streptavidin beads. RAPH1 biotinylation was then assessed by western blotting (representative of three biological repeats).

Fig. 2.

RAPH1 is recruited to filopodia tips in an MYO10-FERM-dependent manner. (A–D) U2-OS cells expressing MYO10–RFP or MYO10^ΔF–RFP together with RAPH1–GFP (A) or VASP–GFP (B) were plated on FN for 2 h, fixed, stained for F-actin and imaged using SIM. (A,B) Representative maximum intensity projection (MIPs) are displayed. Yellow arrows highlight filopodia tips. Scale bars: 20 µm (main); 2 µm (magnifications). (C) Heatmap highlighting the sub-filopodial localization of the proteins imaged in A and B generated from intensity profiles (n>300 filopodia per condition; three biological repeats). (D) The average RAPH1 and VASP staining intensity at filopodia tips measured in B are displayed as box plots. (E,F) U2-OS cells expressing MYO10^WT–RFP or MYO10^ΔF–RFP were plated on FN for 2 h, fixed, stained for F-actin and endogenous RAPH1, and imaged using SIM. (E) A representative ROI is displayed. Yellow arrows highlight filopodia tips. Scale bars: 2 µm. (F) The average intensity of endogenous RAPH1 at filopodia tips is displayed as box plots (n>175 filopodia per condition; three biological repeats). For all panels, the data are shown as dot plots and Tukey boxplots. The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. The P-values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control.

Fig. 3.

RAPH1 is recruited to filopodia tips via a region located after its PH domain. (A) Cartoon representation of RAPH1 domains. The boundaries of the five fragments (F1 to F5) used in this study are highlighted. (B) U2-OS cells expressing MYO10^WT–RFP and one of the four RAPH1 fragments (F1 to F4) were plated on FN for 2 h, fixed, stained for F-actin and imaged using SIM. Representative maximum intensity projection (MIPs) are displayed. Yellow arrows highlight filopodia tips. Scale bars: 10 µm (main); 2 µm (magnifications). (C) The preferential recruitment of the four RAPH1 fragments to filopodia tips (F1 to F4) was assessed by calculating an enrichment ratio (averaged intensity at filopodia tip versus shaft; >415 filopodia per condition, three biological repeats). (D) U2-OS cells expressing MYO10^WT–RFP and the GFP–RAPH1^F2 (GFP-F2) were plated on FN for 2 h, fixed, stained for F-actin, and imaged using SIM. Representative MIPs are displayed. Yellow arrows highlight filopodia tips. Scale bars: 10 µm (main); 2 µm (magnifications). (E) The preferential recruitment of GFP–RAPH1^F2 to filopodia tips was assessed as in C (>427 filopodia per condition, three biological repeats). (F) GFP pulldowns in MDA-MB-231 cells expressing GFP–RAPH1^F2 or GFP alone. Endogenous MYO10 recruitment to the bait proteins was then assessed by western blotting (representative of three biological repeats). (G) Pulldowns using recombinant GST–RAPH1^F2 or GST alone in MDA-MB-231 cell lysates. MYO10 binding to GST–RAPH1^F2 was then assessed by western blotting (representative of three biological repeats). (H) Pulldowns using recombinant GST–RAPH1^F2 or GST alone and recombinant His-tagged MYO10^FERM. MYO10^FERM binding to GST–RAPH1^F2 was then assessed by western blotting (representative of three biological repeats). (I) GFP pulldowns in MDA-MB-231 cells expressing full-length RAPH1–GFP, RAPH1–GFP lacking the F5 fragment (RAPH1^Δ536-587) or GFP alone. Endogenous MYO10 recruitment to the bait proteins was then assessed by western blotting (representative of three biological repeats). The quantifications of MYO10 recruitment to the bait protein are displayed as a SuperPlot where individual repeats are color-coded (P-value calculated using a Welch's t-test). Inputs in F, G and I, 5%. (J,K) U2-OS cells expressing MYO10–RFP together with full-length RAPH1–GFP (J) or RAPH1–GFP lacking the F5 fragment (RAPH1^Δ536-587) were plated on FN for 2 h, fixed, stained for F-actin, and imaged using SIM. (J) Representative MIPs are displayed. Yellow arrows highlight filopodia tips. Scale bars: 10 µm (main); 2 µm (magnifications). (K) The average intensity and preferential recruitment of the two RAPH1 constructs to filopodia tips are displayed as box plots (n>800 filopodia per condition; three biological repeats). For all panels, the data are shown as dot plots and Tukey boxplots (except I). The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. The P-values were determined using a randomization test (except panel I). NS indicates no statistical difference between the mean values of the highlighted condition and the control.

Fig. 4.

RAPH1 supports filopodia formation and the stability of the filopodia tip complex. (A) Efficiency of siRNA-mediated RAPH1 silencing using two different siRNA oligonucleotides in U2-OS cells (representative of three biological repeats). (B) RAPH1-silenced U2-OS cells transiently expressing MYO10–GFP were plated on FN for 2 h, fixed, and the number of MYO10-positive filopodia per cell was quantified (n>93 cells per condition, three biological repeats). Scale bars: 25 µm (main); 5 µm (magnifications). (C) RAPH1-silenced U2-OS cells transiently expressing MYO10–RFP together with GFP, RAPH1^FL–GFP or RAPH1^Δ536-587–GFP were plated on FN for 2 h, fixed, and the number of MYO10-positive filopodia per cell was quantified (n>60 cells per condition, three biological repeats). Scale bars: 25 µm (main); 5 µm (magnifications). (D) RAPH1-silenced U2-OS cells transiently expressing MYO10–GFP were plated on FN, stained for F-actin, and imaged using SIM. Representative maximum intensity projection (MIPs) are displayed. Scale bars: 20 µm (main); 2 µm (magnifications). Quantifications of filopodia length from SIM images are displayed (n>530 filopodia per condition; three biological repeats). (E,F) RAPH1-silenced U2-OS cells transiently expressing MYO10–GFP were plated on FN and imaged live using an Airyscan confocal microscope (1 picture every 5 s over 20 min). For each condition, MYO10-positive particles were automatically tracked. (E) The average MYO10 track duration per cell is displayed (three biological repeats, n>17 cells per condition). (F) The average speed and the total distance traveled by MYO10 spots are displayed (n>9600 filopodia; three biological repeats). For all panels, the data are shown as dot plots (except F) and Tukey boxplots. The whiskers (shown here as vertical lines) extend to data points no further from the box than 1.5× the interquartile range. The P-values were determined using a randomization test. NS indicates no statistical difference between the mean values of the highlighted condition and the control.