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© The Rockefeller University Press, 0021-9525/1997//417 $5.00
The Journal of Cell Biology, Volume 139, Number 2, , 1997 417-434

Article

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



Clare M. Waterman-Storer and E.D. Salmon

Department of Biology, 607 Fordham Hall, University of North Carolina, Chapel Hill, North Carolina 27599-3280

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 µm/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 ~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.

Abbreviations used in this paper: BDM, 2,3-butanedione–2-monoxime; CMTC, cytoplasmic microtubule complex; DIC, differential interference contrast; F-actin, filamentous actin; MT, microtubule; VE, video enhanced.

Address all correspondence to Clare M. Waterman-Storer, Department of Biology, 607 Fordham Hall, University of North Carolina, Chapel Hill, NC 27599-3280. Tel.: (919) 962-2354. Fax: (919) 962-1625. E-mail: waterman @email.unc.edu and tsalmon{at}email.unc.edu

C.M. Waterman-Storer is a Fellow of the Jane Coffin Childs Memorial Fund for Research. This work was supported by National Institutes of Health grant GM 24364 to E.D. Salmon.


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