Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.
We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.
Waterman-Storer
C M
CM
Department of Biology, 607 Fordham Hall, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. waterman@email.unc.edu
Salmon
E D
ED
D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000502:physiology* / Q000648:ultrastructure*; D008168:Lung / Q000166:cytology / Q000502:physiology; D008845:Microinjections; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video / Q000379:methods; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D012455:Salamandridae; D013997:Time Factors; D014404:Tubulin / Q000032:analysis
D014404:Tubulin