Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching.
Following our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses. Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously. Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA-induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin-induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.
Rees
D A
DA
National Institute for Medical Research, Mill Hill, London, United Kingdom.
Charlton
J
J
Ataliotis
P
P
Woods
A
A
Stones
A J
AJ
Bayley
S A
SA
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D003208:Concanavalin A / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000378:metabolism*; D011940:Receptor Aggregation / Q000187:drug effects
D003208:Concanavalin A; D003994:Bucladesine; D009218:Myosins; D002118:Calcium