A novel form of motility in filopodia revealed by imaging myosin-X at the single-molecule level.
Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow, 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. 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 toward the filopodial tip. These fast forward movements occur at approximately 600 nm/s over distances of up to approximately 10 microm 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 toward the filopodial tip, but at a slower characteristic velocity of approximately 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.
Kerber
Michael L
ML
Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Jacobs
Damon T
DT
Campagnola
Luke
L
Dunn
Brian D
BD
Yin
Taofei
T
Sousa
Aurea D
AD
Quintero
Omar A
OA
Cheney
Richard E
RE
D000199:Actins / Q000502:physiology; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000032:analysis / Q000502:physiology* / Q000648:ultrastructure; D011554:Pseudopodia / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D053778:Thiazolidines / Q000494:pharmacology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; D009218:Myosins; C037068:latrunculin B