Figure 8. Filopodia stabilization directs cell migration.

(a) MDA-MB-231 cells transiently expressing mEmerald-Paxillin were plated on FN and imaged live using a TIRF microscope (1 picture every 1 min over 3 h; scale bar, 20 μm) in the presence of DMSO or amlodipine besylate (10 μM). Focal adhesion properties were analysed using the focal adhesion analysis server⁵³ (three biological repeats; over 33 movies per condition analysed; adhesion lifetime and maximal area, n>112,000 adhesions analysed; assembly rate, n>6100 adhesions analysed; disassembly rate, n>7,200 adhesions analysed, ***P value <1.73 × 10⁻⁵⁰). P values were calculated using Student's t-test (unpaired, two-tailed, unequal variance). (b) MDA-MB-231 cells transiently expressing talin-1-GFP and MYO10-mCherry were plated on FN and imaged live using a TIRF microscope (1 picture every 5 s; scale bar, 20 μm). Images of the region of interest (yellow squares) at the time point of interest are displayed on the right. Yellow arrows highlight MYO10-positive filopodia that precede mature focal adhesions. (c) Cartoon representing the sequence of events, identified in this study, leading to filopodia stabilization and ultimately focal adhesion formation. Briefly, unstable filopodia are extended from the plasma membrane to tether the surrounding environment. These filopodia display low calcium concentration at their tips. Molecular motors such as MYO10 transport integrins and other adhesion receptors to filopodia tips. Upon integrin activation, the kinase Src, directly or indirectly, promotes L-type calcium channel activity resulting in increased calcium concentration at filopodia tips. Higher calcium concentration at filopodia tips promotes calpain-1 activation and filopodia stabilization. Ultimately, upon plasma membrane advancement, adhesions initiated at filopodium tips/shafts precede mature classical focal adhesion.