CY, GS, and TS conceived and designed the experiments. CY, LC, SG, and SK performed the experiments. CY, SG, GS, and TS analyzed the data. CY, SK, and GS contributed reagents/materials/analysis tools. TS wrote the paper.
The authors have declared that no competing interests exist.
Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.
Cell motility is a cyclic process, with the protrusion of the leading edge followed by retraction of the rear. Protrusion is driven by polymerization of actin filaments, with the spatial organization of these filaments determining the shape of the protrusions. For example, the spike-like filopodia contain bundles of long actin filaments, whereas the sheet-like lamellipodia contain branched actin networks. In biochemical assays, two stimulators of actin polymerization, Arp2/3 complex and formins, induce branched or individual filaments, respectively. In cells, Arp2/3 complex and formins also appear to be implicated in the formation of lamellipodia and filopodia, respectively. However, when we investigated the role of mDia2 formin by functional approaches, we unexpectedly found that it is essential, not only for filopodia, but also for lamellipodia. Moreover, functions of mDia2 in lamellipodia and filopodia appeared intimately linked. We recorded behavior of cells by light microscopy and then used electron microscopy to study actin architecture in the same cells. We found that an activated form of mDia2 was first recruited to lamellipodia, where it induced many long, unbranched filaments, and from there, drove formation of filopodia through gradual convergence of these lamellipodial filaments into bundles. These data demonstrate a strong relationship between structurally different actin filament arrays and molecular machineries involved in their formation.
Formin mDia2 was believed to function mainly in the generation of filopodia in migrating cells. We unexpectedly found that mDia2 is also important for lamellipodia and induces filopodia in association with lamellipodia.
Cell motility is a cyclic process consisting of protrusion of the leading edge followed by retraction of the rear. Actin filament polymerization provides a driving force for protrusion, whereas the shape and dynamics of protrusive organelles depend on the spatial organization of underlying filaments and activity of accessory molecules. Spatially restricted and temporally controlled actin filament nucleation is critical for generating membrane protrusions. Arp2/3 complex and formins are two major actin filament nucleators acting as convergent nodes of signaling pathways leading to initiation of actin-based motility (reviewed in[
The concept of functional separation of Arp2/3 complex and formins during leading-edge protrusion to generate dendritic networks in lamellipodia and parallel bundles in filopodia, respectively, is challenged by the observation that filopodia may arise by reorganization of the lamellipodial network [
The domain organization of two related formins, mDia1 and mDia2, and functions of individual domains are well characterized [
In this study, aiming to characterize the mDia2-dependent mechanism of filopodia formation, we investigated a role of mDia2 in the leading-edge protrusion by loss-of-function and gain-of-function approaches followed by detailed analyses of the phenotypes using light and electron microscopy (EM) and a combination of both. Unexpectedly, we found that mDia2 was implicated in lamellipodia formation, suggesting that the Arp2/3 complex, despite being essential, was not sufficient for this function. Furthermore, the constitutively active mDia2 mutant efficiently induced filopodia, but these filopodia were formed, not from a focal spot away from lamellipodia, but by the gradual reorganization of lamellipodial filaments converging into filopodial bundles. We also found that specific targeting to the membrane was essential for mDia2 functions in protrusion. The targeting required a multifunctional scaffolding protein Abi1, which also participates in other actin-remodeling events, including regulation of Arp2/3-dependent nucleation [
Mouse melanoma B16F1 cells form broad lamellipodia interspersed by a varying number of filopodia (
(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 (
(D) Inhibition of cell migration by mDia2 siRNA. An asterisk (*) indicates statistical significance (
(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 (
(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).
We investigated mDia2 functions in cells using the RNA interference (RNAi) approach. Transfection of B16F1 cells with mDia2 short interfering RNA (siRNA) significantly depleted endogenous mDia2, but not other proteins involved in actin-based protrusion (
(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
Some filopodia also remained after transfection with mDia2 siRNA, but they appeared less rigid than normal, frequently bending and protruding in a curvy path. Although fascin was present in these filopodia, the number of fascin-containing structures was significantly decreased (
The effects of mDia2 siRNA on lamellipodia and filopodia were specific because they could be rescued by RNAi-resistant full-length green fluorescent protein (GFP)-mDia2 (FL-mDia2*) (
Platinum replica EM was used to reveal specific defects caused by mDia2 depletion in the architecture of the protrusive organelles (
(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
(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).
Collectively, these results reveal a novel role of formin mDia2 in the formation of lamellipodia, likely involving facilitated formation of long filaments, and corroborate its function in filopodial protrusion [
In addition to loss-of-function analysis by RNAi, we also tested mDia2 functions by a gain-of-function approach using a constitutively active mDia2 mutant (ΔGBD-mDia2) lacking GBD and a part of DID [
In B16F1 cells, GFP-ΔGBD-mDia2 localized to a subset of lamellipodia (
(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 (
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
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
(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 (
(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).
Correlative EM analysis ΔGBD-mDia2–positive lamellipodia (
Thus, the gain-of-function approach showed that the constitutively active mDia2 mutant was recruited to lamellipodia and induced structural reorganization of the lamellipodial actin network by promoting formation of long filaments, as well as increased the persistence of lamellipodia protrusion. These results corroborate the findings of mDia2 knockdown experiments, which suggested mDia2 function in lamellipodia.
Our findings of mDia2 involvement in lamellipodia formation apparently contrast with previous reports showing a role of mDia2 [
Filopodia induced by ΔGBD-mDia2 frequently were very long, flexible, not attached to the substratum, and occasionally appeared on the dorsal cell surface (
By EM analysis, ΔGBD-mDia2–mediated filopodia contained actin bundles, which looked normal in their distal domains, but displayed informative differences in the proximal regions (
(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).
Together, these results confirm the previous observations that mDia2 plays a role in filopodia generation, and localizes to filopodial tips. We extend prior observations by showing that although filopodia induced by active mDia2 share many features with normal filopodia, they also display an abnormal abundance of prematurely terminated filaments in the proximal parts of filopodial bundles.
We next asked whether mDia2 roles in lamellipodia and filopodia are related. Kinetic analysis of filopodia initiation showed that virtually all ΔGBD-mDia2–induced filopodia formed from ΔGBD-mDia2–positive lamellipodia (
(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).
To understand the mechanism of lamellipodia-to-filopodia transition, we performed correlative EM for cells with known live history (
The second example (
Correlative analysis of six cells imaged live for 90–120 s showed that all nascent filopodia (31 total) emerged by condensation of linear lamellipodial fluorescence. At EM level, 14 out of these 31 filopodia were similar in structure to the first example in
These data, collectively, indicate that induction of filopodia by active mDia2 temporally and mechanistically follows the mDia2 appearance in lamellipodia. Long, unbranched actin filaments induced by ΔGBD-mDia2 in the lamellipodial network seem to play an important role in this process by expressing a high tendency to converge into bundles, and leading to filopodia induction in association with pre-existing lamellipodia.
FH1FH2 constructs of many formins are sufficient to nucleate actin filaments and bind barbed ends [
(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.
GFP-FH1FH2-mDia2 was not able to rescue the mDia2 siRNA phenotype (
Searching for a molecular mechanism of mDia2 targeting, we concentrated on proteins displaying robust localization to the leading edge of lamellipodia. Ena/VASP proteins were likely not involved, as ΔGBD-mDia2 localized properly in Ena/VASP-deficient MVD7 cells (
Ectopically coexpressed GFP-FL-mDia2 and Abi1 (
(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 [
(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.
To assess the physiological relevance of this interaction, we used a previously characterized stable line of Abi1-RNAi–interfered (Abi1KD) HeLa cells with approximately 90% depletion of Abi1 (
(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 (
(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
Scale bars indicate 10 μm.
We tested next whether the human ortholog of mDia2 (DIAPH3) plays a role in filopodia formation in control and Abi1KD HeLa cells using both loss-of-function and gain-of-function approaches. Similar to B16F1 cells, DIAPH3 siRNA significantly depleted DIAPH3 expression and impaired cell spreading and protrusive activity in control and Abi1KD HeLa cells (
Similar to B16F1 cells, expression of ΔGBD-mDia2 in HeLa cells induced long, robust filopodia (
(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
(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.
In this study, by combination of loss-of-function (RNAi) and gain-of-function (constitutively active mutant) approaches, we investigated a role of mDia2 formin in the actin-based protrusion in motile cells. Although our original goal was to characterize the mechanism of mDia2-dependent filopodia formation, we unexpectedly found that mDia2 is important for lamellipodial protrusion and induces filopodia in association with lamellipodia. Based on our data, we propose a model suggesting that mDia2 is recruited to the lamellipodial leading edge in an Abi1-dependent manner, where it nucleates new filaments and/or maintains filament elongation by protecting barbed ends from capping. In the course of elongation, lamellipodial filaments converge and become bundled, generating filopodia.
We found that mDia2 can be recruited to the leading edge by at least two mechanisms, one of which relies on binding of the mDia2 FH1FH2 module to barbed ends and another on N-terminal mDia2 sequences interacting with Abi1. The FH1FH2 module contributes to localization to filopodial tips by binding to and riding on elongating barbed ends, which accumulate at the membrane after encountering the boundary conditions [
The majority of Abi1 has been shown to be engaged in complex with WAVE, Nap1, and PIR121 [
Specific targeting of proteins is a common way to confine the protein activity to a certain area. Therefore, mDia2 targeting to lamellipodia and filopodia points to an idea that mDia2 may function there. Although mDia2′s role in filopodia is expected [
Biochemical activities of mDia2 suggest that it may contribute to lamellipodia formation through nucleation and/or anticapping protection of actin filaments (
(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.
It is unclear why mDia2 has such a significant role in lamellipodial protrusion, considering the well-established involvement of Arp2/3 complex in this process [
A role of mDia2 in filopodia formation in mammalian cells [
The overall process of filopodia initiation by mDia2 was very similar to that described for naturally emerging filopodia [
Because of formation of atypical club-like filopodia, we suppose that ΔGBD-mDia2 does not fully recapitulate the physiological pathway of filopodia formation. Based on the structural data, we previously proposed that Arp2/3 complex nucleates filaments for filopodial bundles [
In summary, we have shown that mDia2 plays a role in formation of both lamellipodia and filopodia. However, these roles are not separate, but related to each other; mDia2 first participates in lamellipodia formation and then induces filopodia from lamellipodia. A most likely scenario is that mDia2-nucleated actin filaments and/or filaments nucleated by Arp2/3 complex and protected by mDia2 are initially dispersed in the lamellipodial network, and subsequently, they elongate persistently and gradually converge, eventually segregating themselves into filopodial bundles. Although regulation of actin dynamics is the most straightforward way to explain how mDia2 may function in protrusion, we cannot exclude a possibility that other mDia2 activities, such as regulation of microtubule dynamics [
B16F1 mouse melanoma cells [
Samples for platinum replica EM were processed as described [
All morphometric measurements were done using MetaMorph Imaging software (Molecular Devices) unless stated otherwise. Graphs and statistical analysis were done using SigmaPlot software. Statistical significance was determined by the Student
Quantification of lamellipodia and filopodia were performed as described [
For spreading assays, the projected cell area was measured 2 h after plating. For cell migration analysis, cells transfected with mDia2 siRNA (Cy3 labeled) or control siRNA (nonlabeled) were co-cultured in 35-mm dishes and imaged 36–48 h post-transfection (4 h after plating). Phase contrast time-lapse sequences were acquired with 5-min intervals for 6 h using a 4× objective. Fluorescence images to identify siRNA-transfected cells were acquired immediately before and after each movie. Cell positions were recorded every ten frames using Track Object tool in MetaMorph. An average instantaneous rate was calculated for each cell, and then the mean speed of cell migration was determined for each group of cells. To analyze the lamellipodial dynamics, time-lapse sequences were acquired with 3-s intervals for 10 min using a 100× objective. Kymographs were generated along straight lines drawn in the direction of protrusion. Rates of lamellipodia protrusion were determined based on slopes produced by advancing leading edges. Persistence corresponds to time intervals during which individual protrusions occurred. The relative mDia2 levels in control and mDia2 siRNA-treated cells were determined by immunostaining after fixation with paraformaldehyde and permeabilization with Triton X-100. Integrated fluorescence intensity of mDia2 staining for each cell after background subtraction was plotted against the fraction of the cell edge occupied by lamellipodia.
To determine the degree of mutual orientation of actin filaments in lamellipodia of control and ΔGBD-mDia2-expressing cells, square EM images of the lamellipodial network ranging from 0.13 to 0.53 μm2 were thresholded using Adobe Photoshop to maximally highlight the linear features. Matlab software was used to generate two-dimensional Fast Fourier Transform from these images, collect radial intensity line scans, plot intensity as a function of angle, and fit a Gaussian curve to the major peak. Standard deviation of the Gaussian fit was used as a parameter of the orientational order. Matlab code for this analysis was provided by J. Winer, Q. Wen, and P. Janmey (University of Pennsylvania).
For immunoblotting, cells were lysed in buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, and a protease inhibitor tablet (Roche). Protein concentration of the lysates was determined using Bio-Rad protein assay kit (Bio-Rad). Proteins were separated by SDS-PAGE (7.5%–10% polyacrylamide). Tubulin was used as loading control. Immunoblots were developed using ECF Western blotting kit (Amersham).
Coimmunoprecipitation and in vitro binding assays were performed as described [
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Cell populations transfected with mDia2 siRNA (A) or cotransfected with mDia2 siRNA and siRNA-resistant GFP-FL-mDia2* or GFP-RacV12 (B). mDia2 knockdown inhibits lamellipodia; this phenotype can be rescued by FL-mDia2*, but not by GFP-Rac1V12. Bars indicate 25 μm.
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Distribution of endogenous mDia2 (red) and Abi1 (green) in lamellipodia of B16F1 cell, as detected by immunostaining. Bar indicates 5 μm.
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Both unbound and branched proximal (“pointed”) ends of actin filaments can be detected in ΔGBD-mDia2–induced lamellipodia.
Top: anaglyph stereo image (right eye blue) showing 3D organization of filaments in ΔGBD-mDia2–induced lamellipodia.
Bottom: 2D image of the same region with unbound ends marked by yellow dots, and ends engaged in branch formation by red dots. Boxed region is enlarged in the inset with branched filaments highlighted in blue. Scale bar indicates 0.2 μm.
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Examples show formation of a club-like filopodium (arrowhead) and a dorsal filopodium (arrow).
Top: phase contrast.
Middle: GFP fluorescence in inverse contrast.
Bottom: overlay with GFP in red.
Arrowhead points to the formation of a club-like filopodium by fusion of several smaller filopodia. Discontinuous linear fluorescence of ΔGBD-mDia2 (0:00 time point) gradually converges (0:30) and produces two distinct dots at the tips of small filopodia (1:00). Another filopodium is seen in-between with barely detectable GFP signal. The right filopodium moves laterally (1:00 through 2:00), and all three filopodia fuse (2:30), producing a single filopodium that protrudes extensively and acquires a club-like shape.
Arrow points to the formation of a dorsal protrusion from a lateral filopodium. Linear fluorescence at the lower right side of the lamellipodium (0:30) gradually produces two filopodia by the 2:00 time point, which fuse (3:00), and the resulting structure translocates to the dorsal surface of lamella.
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(A) Phenotype induced by expression of GFP-ΔN2-mDia1. GFP fluorescence of GFP-ΔN2-mDia1 (left) and F-actin enrichment (middle) are found throughout the cytoplasm of a bipolar cell as previously described in other cells [
(B) Expression of GFP-tagged mDia2 constructs in B16F1 cells. FH1 domain, residues 519–600 (FH1), and truncated FH1FH2, residues 519–909 (trFH1FH2) have cytoplasmic distribution and do not induce filopodia. Scale bars indicate 10 μm.
(C) Filopodia induction does not depend on Ena/VASP proteins. Expression of GFP-ΔGBD-mDia2 (a and b) or mRFP1-ΔGBD-mDia2 (c) in Ena/VASP-deficient MVD7 cells (a) or MVD7 cells stably re-expressing GFP-Mena (MVD7-EM) (b) or transiently re-expressing GFP-VASP (c). (a and b) ΔGBD-mDia2 localizes to the membrane and induces filopodia equally well in MVD7 and MVD7-EM cells. (c) In cells expressing relatively low levels of ΔGBD-mDia2 (top row), re-expressed VASP is still occasionally present at filopodial tips (arrow), but is displaced to more proximal regions of filopodia (arrowheads) in highly expressing cells (bottom row). Scale bars indicate 5 μm in (a and b) and 10 μm in (c).
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(A) Western blotting of lysates from control or Abi1KD HeLa cells. Amount of protein loaded is shown in μg. Expression of Abi1 is decreased by approximately 90% in Abi1KD cells, but Arp2/3 subunit p34-Arc (p34) is not changed.
(B) EM of a peripheral region of a control HeLa cell.
(C) EM of a peripheral region of Abi1KD cell with two filopodia. Boxed regions showing branched filaments in filopodial roots are enlarged at the bottom as 3D anaglyph images (right eye red) (top row), and as 2D images with branched filaments highlighted in color (bottom row). Although lamellipodia are grossly inhibited in these cells, small regions of dendritic network can be occasionally detected at cell edges (arrow).
Bars indicate 0.5 μm.
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B16F1 cell at the left is transfected with mDia2 siRNA and has very low protrusive activity. Control untransfected B16F1 cell at the right forms lamellipodia and filopodia. This video corresponds to
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Lamellipodia and filopodia are severely inhibited.
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Expression of GFP-ΔGBD-mDia2 (red) induces formation of dynamic filopodia in a B16F1 cell. GFP-ΔGBD-mDia2 remains associated with filopodial tips during protrusion. This video corresponds to
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Coexpression of GFP-actin (white) and mRFP1-ΔGBD-mDia2 (not shown) in B16F1 cell shows actin enrichment at the thicker termini of GFP-ΔGBD-mDia2–induced filopodia. Arrow points to an actin speckle which undergoes slow retrograde flow and finally disappears, while the filopodial tip protrudes forward. This behavior illustrates actin assembly at the tip, whereas disappearance of the speckles and overall decrease of actin intensity toward the rear indicates actin disassembly in proximal regions. This video corresponds to
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Linear fluorescence along the lamellipodial leading edge gradually transforms into a series of bright dots at the filopodial tips. This video corresponds to
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Linear fluorescence along the lamellipodial leading edge converges into dots at the filopodial tips (arrows).
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Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to
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Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to
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Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to
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The time-lapse sequence of B16F1 cell expressing GFP-FH1FH2-mDia2. GFP-FH1FH2-mDia2 displays strong cytoplasmic localization and is mildly enriched at the tips of dynamic filopodia.
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We thank Drs. A. Alberts, R. Cheney, F. Gertler, H. Higgs, K. Kaibuchi, S. Narumiya, D. Schafer, R. Tsien, and T. Uruno for generous gifts of reagents, P. Sterling for permission to use his JEOL 1200EX electron microscope, S. H. Zigmond and G. G. Borisy for fruitful discussions, and J. Winer and P. Janmey for help with quantification of filament orientation.
Abi1-RNAi-knockdown cells
green fluorescent protein
RNA interference
short interfering RNA
WAVE/Abi1/Nap1/PIR121/HSP300 complex