Simultaneous stretching and contraction of stress fibers in vivo.
To study the dynamics of stress fiber components in cultured fibroblasts, we expressed alpha-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and alpha-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and alpha-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.
Peterson
Lynda J
LJ
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA. ljpeters@med.unc.edu
Rajfur
Zenon
Z
Maddox
Amy S
AS
Freel
Christopher D
CD
Chen
Yun
Y
Edlund
Magnus
M
Otey
Carol
C
Burridge
Keith
K
D000185:Actinin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008246:Lysophospholipids / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D018994:Myosin Light Chains / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D024462:Myosin Type II / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D038761:Photobleaching; D022502:Stress Fibers / Q000502:physiology* / Q000648:ultrastructure*; D041701:Swiss 3T3 Cells
D008246:Lysophospholipids; D008387:Marine Toxins; D018994:Myosin Light Chains; D010080:Oxazoles; C485184:enhanced green fluorescent protein; D000185:Actinin; D049452:Green Fluorescent Proteins; C059041:calyculin A; D024462:Myosin Type II; C032881:lysophosphatidic acid