Quantitation of cytoskeletal fibers in fluorescence images: Stress fiber disassembly accompanies dephosphorylation of the regulatory light chains of myosin II

Abstract

Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the assembly dynamics of the actin-based cytoskeleton. Previous studies suggested that fibers containing actin and myosin II are restructured via two alternative pathways: coupled solation-contraction and disassembly without contraction. The former process appears to be regulated by Ca++, but regulation of the latter process has not been extensively explored. To assess the possible role of protein phosphorylation, we examined the dynamic behavior of stress fibers in cells treated with inhibitors of protein kinases. Swiss 3T3 fibroblasts were microinjected with fluorescent analogs of actin and myosin II and fiber dynamics monitored using light-microscope imaging. Staurosporine and KT5926 caused reversible dispersal of stress fibers without contraction, along with dephosphorylation of the regulatory light chain of myosin II, LC20. In order to make direct comparisons between the dose responses of these biochemical and morphological effects, fiber disruption was quantitated using two independent measures: total edge strength in fluorescent images of actin and myosin II, and fiber length determined by automated object-identification. Loss of stress fibers was shown to parallel LC20 dephosphorylation. Quantitation of cytoskeletal organization provides a framework for testing relationships between structural events and potential biochemical regulatory signals.