A Novel Form of Motility in Filopodia Revealed by Imaging Myosin-X at the Single-Molecule Level

Abstract

Summary Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow[1-3], it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia[4]. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion towards the filopodial tip. These fast forward movements occur at ∼600 nm/s over distances of up to ∼10 μm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction-limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move towards the filopodial tip, but at a slower characteristic velocity of ∼250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…2 A and B and Movies S1 and S2). The speeds are similar to those measured for EGFP labeled myosin-10 molecules tracked within filopodia by live cell fluorescence imaging, reported as 580 and 840 nm/s at 25°C and 37°C, respectively (20).…”

Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics

“…8). This was similar to the rearward movement of Myo10, corresponding to retrograde flow (33,34). Furthermore, the fluorescence intensity of retreating spots was strong, suggestive of complex formation.…”

Antiparallel coiled-coil–mediated dimerization of myosin X

“…4 A5 and C1), indicating that anti-CC-mediated dimerization is critical for Myo10's processive movement and localization to the filopodial tip. This GCN4-substituted Myo10 head still showed significantly more filopodial tip puncta than anti-CC-disruption mutants, consistent with previous reports showing that the forced parallel coiled-coil dimerization of the Myo10 head can trigger filopodial tip localization (15,21). To further assay the anti-CC's function in Myo10-induced filopodial induction, we counted elongated filopodial numbers in both HeLa and COS7 cells using the GFPtagged full-length Myo10.…”

“…1A). Both in vitro single-molecule-based experiments and in vivo near single-molecule imaging assays have shown that Myo10 is capable of processive movements along actin filaments; thus it is expected that Myo10 can dimerize (15,16). Additionally, the role that Myo10 plays in filopodial induction suggests that it may function as a bundled actin filament-selective motor (17,18).…”