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. 2014 Sep 1;25(17):2604–2619. doi: 10.1091/mbc.E14-02-0712

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© 2014 Barzik et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Microtubule targeting of mDia2^M/A-initiated filopodia. (A) mDia2^M/A-assembled filopodia contain microtubules. MV^D7 cells were transiently transfected, chemically fixed, and probed for microtubules (α-tubulin), filopodia (Lpd), and F-actin (phalloidin). Arrows indicate microtubules extending into tips of mDia2^M/A-assembled filopodia. Arrowheads show that microtubules do not extend into filopodia of control or GFP-VASP–expressing cells. (B) Actively polymerizing microtubules perpetually protrude into mDia2^M/A-induced filopodia. Montages of single frames from 3-min time series of cells coexpressing mCherry-EB1 and either GFP-VASP (top) or GFP-mDia2^M/A (bottom). Arrowheads indicate EB1-labeled microtubules actively entering mDia2^M/A-labeled filopodia. (C) Filopodia in GFP-VASP–expressing (left) or GFP-mDia2^M/A–expressing (right) cells are unaffected by microtubule depolymerization. Transfected cells were exposed to 100 ng/ml nocodazole for 15 h (DMSO control, top; nocodazole, bottom) and probed for α-tubulin and F-actin (phalloidin). Nocodazole does not affect filopodia formation in GFP-VASP–expressing cells (filled arrowheads). DMSO treatment does not prevent microtubules from entering GFP-mDia2^M/A–labeled filopodia (arrows). Depolymerization of microtubules does not inhibit filopodia formation in cells expressing GFP-mDia2^M/A but reduces filopodial length (open arrowheads). (D–F) Quantification of the experiments shown in A and C. (D) mDia2^M/A-labeled filopodia do not contain microtubules in nocodazole-treated cells. (E) Microtubules are not required for GFP-mDia2^M/A–driven filopodia formation. (F) Microtubule depolymerization dramatically reduces filopodia length in cells expressing GFP-mDia2^M/A, whereas filopodial length in GFP-VASP–expressing cells is slightly increased. Data in D–F represent at least two repetitions. *p < 0.05; ***p < 0.001. DMSO, vehicle control; nocod., nocodazole. n = 39–55 cells. Scale bars, 5 μm.