Figure 1.
Depletion of mDia2 in B16F1 Cells by siRNA
(A) Staining of endogenous mDia2 by antibody (red) and actin by phalloidin (green). mDia2 is concentrated in lamellipodia and at filopodial tips (see insets at right).
(B) Western blotting of cell populations transfected with control or mDia2 siRNA. Tubulin is used as a loading control. Amount of mDia2 is decreased by approximately 60%; amounts of other proteins involved in protrusion do not decrease.
(C) Inhibition of cell spreading by mDia2 siRNA. Projected cell area was determined 2 h after cell plating. Top and bottom of a box indicate 75th and 25th quartiles, respectively; whiskers indicate 10th and 90th percentiles, respectively; dot is the mean; and the middle line is the median. An asterisk (*) indicates statistical significance (p < 0.0001, n = 120–129 cells).
(D) Inhibition of cell migration by mDia2 siRNA. An asterisk (*) indicates statistical significance (p < 0.001, n = 34–35 cells).
(E) Inhibition of lamellipodia protrusion by mDia2 siRNA. Analysis of kymographs (top) showed that rate and persistence of lamellipodia (bottom) are reduced in mDia2 siRNA-treated cells (red) compared to control siRNA (blue). Coexpression of siRNA-resistant GFP-mDia2* (purple) partially rescues the rate and fully rescues the persistence of mDia2 siRNA cells. Differences between datasets connected by brackets are statistically significant (p < 0.001, n = 20–36 cells). Red arrows in kymographs indicate the direction and the scale of time and distance.
(F) Cell transfected with mDia2 siRNA (left), in contrast to control cell (right), poorly forms lamellipodia. Numbered boxes indicate regions for the time-lapse sequences shown in (G).
(G) Frames from time-lapse sequence (25 s apart) for boxed regions in (F) (top, box 1 is shown; middle, box 2; and bottom, box 3).
(H) Immunostaining of p16-Arc subunit of Arp2/3 complex (left) and fascin (right) in control and mDia2 siRNA-treated cells. Both proteins lose their characteristic enrichment in lamellipodia and filopodia, respectively. mDia2 siRNA is shown in red.
Scale bars indicate 10 μm in (A); 10 μm in (F); and 25 μm in (H).
Figure 2.
Rescue of mDia2 siRNA Phenotype
(A) Phalloidin staining (white) in cells transfected, as indicated, with control or mDia2 siRNA (red) and cotransfected with siRNA-resistant GFP-FL-mDia2*, GFP-mDia1, GFP-Rac1V12, GFP-VASP, or GFP-FH1FH2-mDia2 (green), as indicated. Lamellipodia formation is inhibited by mDia2, but not by control siRNA, and can be rescued by GFP-FL-mDia2*, but not by other proteins. Cyan lines mark examples of edges scored as lamellipodia during quantification shown in (B). Insets in phalloidin channels show close-ups of cell edges scored as positive (1) of negative (2) for lamellipodia presence according to the criteria described in Materials and Methods. Inset in GFP-FL-mDia2 panel shows FL-mDia2* at filopodial tips in another cell.
(B and C) Quantification of lamellipodia (B) and filopodia (C) expression in conditions shown in (A). Box-and-whisker plots are as in Figure 1. All differences are statistically significant. n = 21–38 cells. Scale bars indicate 10 μm.
Figure 3.
EM of Protrusions in Control (A and B) and mDia2-Depleted (C–G) B16F1 Cells
(A and B) Overview of an edge (A) and enlargement of the boxed region (B) of untreated cell show dense actin filament network in lamellipodia and parallel bundles in filopodia. Filaments in filopodia roots splay apart and merge with the surrounding network (A). Brackets in (A) mark a region analyzed in Figure 5E.
(C) Overview of mDia2-depleted cell. Boxes indicate the magnified regions shown in (D) and (E).
(D and E) Intermediate magnification of boxed regions in (C). Boxes indicate the magnified regions shown in (F) and (G).
(F and G) High magnification of boxed regions in (E) and (D), respectively. Note overall inhibition of protrusions (C–E) and abnormal organization of remaining lamellipodia (F) and filopodia (G).
Scale bars indicate 1 μm in (A), (D), and (E); 0.2 μm in (B), (F), and (G); and 5 μm in (C).
Figure 4.
Lamellipodia of ΔGBD-mDia2–Expressing B16F1 Cells
(A) Distribution of GFP-ΔGBD-mDia2 (red) and indicated cytoskeletal proteins (green) detected by phalloidin (Actin) or immunostaining (all others). ΔGBD-mDia2–positive lamellipodia contain normal lamellipodial components: actin, Arp2/3 complex, capping protein, VASP, Abi1, and a filopodial marker, fascin. Scale bars indicate 2.5 μm.
(B) Lamellipodia dynamics in control and ΔGBD-positive lamellipodia. Left: kymographs; right: quantification of the rate and persistence of lamellipodial protrusion. ΔGBD-positive lamellipodia are slower, but remarkably persistent, as compared to control cells (n = 23–39 cells, p < 0.0001). Red arrows in kymographs indicate the direction and the scale of time and distance.
Figure 5.
Structural Organization of Lamellipodia in ΔGBD-mDia2–Expressing Cells
Correlative EM is used to unambiguously identify ΔGBD-mDia–positive regions.
(A) Phase contrast image overlaid with the GFP-ΔGBD-mDia2 image in red.
(B) Low-magnification EM image of the periphery of the same cell overlaid with GFP image in red. Boxes indicate the magnified regions shown in (C) and (D).
(C and D) Enlarged boxed regions from (B). (C) ΔGBD-mDia2–negative lamellipodium containing branching actin network.
(D) Lamellipodium with continuous GFP fluorescence is dominated by densely packed, long parallel filaments. Box indicates a region analyzed in Figure 5F.
Scale bars indicate 10 μm in (A) and in 0.5 μm (C) and (D).
(E–G) Actin filament orientation in control and ΔGBD-mDia2–positive lamellipodia. (E and F) Plots of radial intensity versus angle (blue) of Fourier transforms generated from the ΔGBD-mDia2–positive lamellipodium in the box in (D) (E), and from the control lamellipodium indicated by brackets in Figure 3A (F). Standard deviation (σ) of Gaussian fits (red), which is used as a parameter of mutual filament orientation, is shown in respective plots. (G) Distributions of σ for ΔGBD-mDia2-positive and control lamellipodia are significantly different (p < 0.0001; n = 8–9 cells).
Figure 6.
Filopodia Induction by ΔGBD-mDia2
(A) Cytoskeletal proteins in ΔGBD-induced filopodia. GFP-, YFP-, or mRFP1-ΔGBD-mDia2 (red)-induced filopodia contain filopodial markers, actin, fascin, VASP, and myosin X, but Abi1 is not enriched there. Fascin, which normally localizes throughout the filopodia, is present only in thick distal domains. VASP localizes to filopodia tips (panel CFP-VASP) or shafts (VASP) in cells expressing low and high levels of ΔGBD-mDia2, respectively. Myosin X at the filopodial tips either colocalizes with or is more distal than GFP-ΔGBD-mDia2. Indicated proteins were visualized by coexpressing fusion proteins (panels CFP-VASP and GFP-myosin X), phalloidin staining (Actin), or immunostaining (all others).
(B) Control B16F1 cell contains relatively short filopodia partially or completely embedded into lamellipodia. Arrow indicates a filopodium shown in (D).
(C) Cell expressing GFP-ΔGBD-mDia2 has long, curvy filopodia with ΔGBD-mDia2 at their tips. GFP fluorescence alone (left) and phase overlaid with GFP image in red (right). Arrow indicates a filopodium shown in (D).
(D) Filopodia dynamics. Frames from time-lapse sequences showing protrusion of control filopodium (top) and ΔGBD-mDia2–induced club-like filopodium (bottom) indicated by arrows in (B) and (C), respectively. Time shown in minutes:seconds.
(E) Protrusion rates of control (n = 48) and ΔGBD-mDia2–induced (n = 62) filopodia. Box-and-whisker plots are as in Figure 1. The difference is statistically significant at p < 0.001.
(F–H) Dynamics of GFP-actin in mRFP-ΔGBD-mDia2–expressing cell. Overview (F) shows filopodia having actin (green) enriched in distal segments and mRFP-ΔGBD-mDia2 (red) localizing at their tips. Boxed filopodium is enlarged at right. Time-lapse sequence (G) and kymograph (H) of the boxedfilopodium from (F) shows retrograde movement and disappearance of actin speckles (arrowheads) while the filopodium protrudes.
Scale bars indicate 2.5 μm in (A); 10 μm in (B) and (C); 1 μm in (D); or 2.5 μm in (F).
Figure 7.
EM of ΔGBD-mDia2–Positive Filopodia
(A) Overview of a peripheral region of ΔGBD-expressing cell. Arrows point to filopodia enlarged in insets.
(B) Club-like filopodium induced by ΔGBD-mDia2. Numerous filaments present in the thick terminal bundles are gradually lost as the bundle tapers towards the rear (bottom of the image). Boxed region in left panel is enlarged at right, and unbound filament ends are marked by arrows.
(C) Filopodium in ΔGBD-mDia2–expressing cell contains filaments originating from branch points (lower insets, wide arrows) and filaments having unbound “pointed” ends (upper inset, narrow arrows).
Scale bars indicate 0.5 μm in (A); 0.2 μm on (B); or 0.1 μm in (C).
Figure 8.
Initiation of ΔGBD-mDia2–Induced Filopodia in B16F1 Cells
(A) Time-lapse sequence shows how linear lamellipodial distribution of GFPΔGBD-mDia2 transforms into a series of dots. Time is shown in minutes:seconds.
(B–J) Correlative EM of nascent ΔGBD-mDia2–induced filopodia.
(B) Phase contrast of ΔGBD-mDia2–expressing cell overlaid with GFP image in red (left) and frames from live GFP sequence including an image after extraction (Ext) (right). Most fluorescence remains after extraction.
(C) EM overview of the periphery of the cell shown in (B) overlaid with the GFP image taken after extraction (red). Boxes indicate regions shown in (D) and (E) (left) and in (F) and (G) (right).
(D–G) GFP (D) and (F) and phase overlaid with GFP in red (E) and (G) sequences showing formation of nascent filopodia. In both cases, GFP dots at the tips of filopodia (arrows) were formed by condensation of linear lamellipodial fluorescence (brackets).
(H–J) EM of nascent filopodia shown in (D) and (E) ([H]) or in (F) and (G) ([I] and [J]). Colors in (H) and (I) represent projected GFP images from corresponding regions taken at those time points: yellow, 0:00; red, 0:50; and green, 1:30. Since ΔGBD-mDia2 is dynamically associated with the advancing cell edge, its localization marks the position of the cell edge at the respective time point. Therefore, only structures behind the color line existed at that time, and structures in front of the line were formed later. In (H), ΔGBD-mDia2 fluorescence at the 0:00 time point (yellow) projects to a region with unbundled filaments behind it; subsequently, filaments begin to converge, and a partially condensed fluorescence (red, 0:50) projects to partially bundled filaments; by the end of the sequence, when GFP condenses into a dot (green), a bundle is formed. In (I), line of ΔGBD-mDia2 in the first frame (yellow) projects to a region with sparse actin filaments, possibly due to network disassembly (see text).
Scale bars indicate 5 μm in (A) and (B); 1 μm in (C); or 0.5 μm in (H–J).
Figure 9.
Targeting of mDia2 to the Leading Edge
(A) GFP-FH1FH2-mDia2 in B16F1 cells is not efficiently targeted to the membrane and weakly induces filopodia. GFP fluorescence (top) and F-actin (middle) are enriched throughout the cell. Boxed region is enlarged at right. Contrast enhancement shows some FH1FH2-mDia2 at the tips of filopodia.
(B) FH1FH2-mDia2, in contrast to ΔGBD-mDia2, is not anchored to the cytoskeleton. Cells expressing GFP-FH1FH2-mDia2 (top) or ΔGBD-mDia2 (bottom) are shown live (left) and detergent-extracted (right) after identical image processing.
Figure 10.
(A) mDia2 and Abi1 coimmunoprecipitate. Total cellular lysates (1 mg) of 293T cells cotransfected with GFP-FL-mDia2 and Abi1, alone or in combination, were immunoprecipitated with an anti-Abi1 antibody. Lysates (20 μg) and immunoprecipitates (IP) were immunoblotted (IB) with the indicated antibodies. Slower migrating Abi1 bands reflect hyperphosphorylation associated with ectopic expression of Abi1 [11,49].
(B) mDia2 and WAVE2 do not coimmunoprecipitate. Total cellular lysates (1 mg) of 293T cells cotransfected with GFP-FL-mDia2, WAVE2, and Abi1, alone or in combination, were immunoprecipitated with an anti-WAVE2 antibody. Lysates (20 μg) and immunoprecipitates (IP) were immunoblotted (IB) with the indicated antibodies.
(C) Recombinant full-length Abi1 binds FL-mDia2. Total cellular lysates (1 mg) of 293T cells, untransfected (control) or transfected with GFP-FL-mDia2 (mDia2), were incubated with 1.5 μM of immobilized GST-Abi1 or GST. Lysates (20 μg) and bound proteins were resolved by SDS-PAGE, stained with Ponceau S to detect GST-fusion proteins, and immunoblotted (IB) with the indicated antibodies.
(D) The SH3 domain-containing fragment of Abi1 mediates the interaction with mDia2. Total cellular lysates (1 mg) of 293T cells, untransfected or transfected with GFP-FL-mDia2 (FL), GFP-FH1-mDia2 (FH1), GFP-FH1FH2-mDia2 (FH1FH2), GFP-ΔGBD-mDia2 (ΔGBD), or GFP-N-terminal fragment of mDia2 (NT) were incubated with 1.5 μM immobilized GST-Abi1-SH3 (SH3-Abi1; aa 330–480) or GST. Lysates (20 μg) and bound proteins were resolved by SDS-PAGE, stained with Ponceau S to detect GST-fusion proteins and immunoblotted (IB) with the indicated antibodies.
Molecular weight markers in (C) and (D) are shown in blue.
(E) Summary of binding activities of mDia2 constructs to SH3-Abi1.
Figure 11.
Roles of Abi1 and DIAPH3 in Protrusion
(A) Inhibition of protrusions by DIAPH3 siRNA (phalloidin staining). Boxed regions are shown enlarged at the bottom of the panel.
(B) Western blot of Abi1KD and control HeLa cells after transfection with control or DIAPH3 siRNA. Abi1KD cells express 75% DIAPH3 compared to control HeLa cells. DIAPH3 siRNA depleted 80% and 55% of DIAPH3 in control and Abi1KD cells, respectively.
(C) Inhibition of spreading of control and Abi1KD cells by DIAPH3 siRNA. Projected cell area is determined 2 h after cell plating. Differences between datasets connected by brackets are statistically significant (p < 0.0001, n = 130–252 cells).
(D) and (E) Filopodia number (D) and length (E) in Abi1KD and control cells after transfection with control or DIAPH3 siRNA. Box-and-whisker plots are as in Figure 1. Differences between datasets connected by brackets are statistically significant (p < 0.001, n = 265–1,102 filopodia from 11–31 cells).
Scale bars indicate 10 μm.
Figure 12.
Phenotype of ΔGBD-mDia2 Expression in Abi1KD Cells
(A) GFP-ΔGBD-mDia2 in Abi1KD cells (top row) is not efficiently targeted to the membrane and weakly induces filopodia, as compared to control HeLa cells (bottom row). GFP is shown in the left column and phase contrast overlaid with GFP in red in the middle column. Filopodia indicated by arrows are identically enlarged (approximately 3-fold) in insets at right.
(B and C) Filopodia number (B) and length (C) in Abi1KD and control HeLa cells after transfection with ΔGBD-mDia2. Box-and-whisker plots are as in Figure 1. Differences between datasets connected by brackets are statistically significant (p < 0.001, n = 265–1,102 filopodia from 11–31 cells).
(D) GFP-ΔGBD-mDia2 in Abi1KD cells is more sensitive to detergent extraction than in control HeLa cells. Abi1KD (top) or control (bottom) cells expressing GFP-ΔGBD-mDia2 are shown live (left) or detergent-extracted (middle and right) after identical image processing (middle) or contrast-enhanced to better visualize tips of filopodia (right).
Scale bars indicate 10 μm.
Figure 13.
Models for mDia2 Functions in Lamellipodia and Filopodia
(A) In lamellipodia, mDia2 is targeted to the membrane in an Abi1-dependent manner, where it may nucleate “mother” filaments, which serve as a base for Arp2/3-dependent nucleation, and/or protect from capping elongating barbed ends, which are nucleated by mDia2 itself or by Arp2/3 complex. Other barbed ends may be protected from capping by VASP. Unnecessary barbed ends are capped by capping protein.
(B) During formation of filopodia, lamellipodial filaments associated at their barbed ends with mDia2 or VASP converge during elongation and become cross-linked, thus forming filopodial bundles.