Figure S1: The Correlation of the Intensity of mDia2 Immunostaining with Lamellipodia ExpressionEach data point represents an individual cell.a.u., arbitrary units.
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Figure S2: mDia2 Knockdown and Its Rescue by FL-mDia2*, but Not by Rac1V12
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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Figure S3: Immunostaining of mDia2 and Abi1
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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Figure S4: The 3D Structure of ΔGBD-mDia2–Induced Lamellipodia
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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Figure S5: Dynamics of Filopodia Formation in B16F1 Cells Expressing ΔGBD-mDia2
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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Figure S6: Phenotypes Induced by Expression of ΔN2-mDia1 or Inactive mDia2 Mutants in B16F1 Cells and by ΔGBD-mDia2 in Ena/VASP-Deficient Cells
(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 [43
]. Only very short, finger-like protrusions with slight enrichment of ΔN2-mDia1 at the tips could be observed at the cell edges abutted by actin bundles (right). No filopodial-like protrusions reaching significant length were observed. Arrow in the merged panel points to a region enlarged at right.
(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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Figure S7: Characterization of Abi1KD HeLa Cells
(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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Video S1: Inhibition of Protrusive Activity of B16F1 Cell Transfected with mDia2 siRNA
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 F.
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Video S2: Another Example of B16F1 Cell Transfected with mDia2 siRNA
Lamellipodia and filopodia are severely inhibited.
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Video S3: Formation of Dynamic Filopodia by GFP-ΔGBD-mDia2
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 C.
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Video S4: Kinetics of GFP-Actin during Filopodia Formation Induced by mRFP-ΔGBD-mDia2 in B16F1 Cell
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 F–H.
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Video S5: Kinetics of GFP-ΔGBD-mDia2 during Filopodia Formation in B16F1 Cells
Linear fluorescence along the lamellipodial leading edge gradually transforms into a series of bright dots at the filopodial tips. This video corresponds to A.
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Video S6: Kinetics of GFP-FL-mDia2 during Filopodia Formation in B16F1 Cells
Linear fluorescence along the lamellipodial leading edge converges into dots at the filopodial tips (arrows).
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Video S7: GFP-ΔGBD-mDia2 Expressing B16F1 Cell Used for Correlative EM with Known History
Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to B.
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Video S8: Formation of a Nascent Filopodium (~30 second old) in GFP-ΔGBD-mDia2–Expressing B16F1 Cell
Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to D and E.
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Video S9: Formation of a Nascent Filopodium (~90 second old) in GFP-ΔGBD-mDia2–Expressing B16F1 Cell
Top: fluorescence; bottom: phase overlaid with fluorescence in red. This video corresponds to F and G.
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Video S10: Kinetics of GFP-FH1FH2-mDia 2 in B16F1 Cell
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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