Calcium/calmodulin induces phosphorylation of vimentin and myosin light chain and cell rounding in cultured adrenal cells.
In previous reports on adrenal cells we have shown that calcium/calmodulin regulates cholesterol transport to mitochondria and induces phosphorylation of cytoskeleton homogenates. In this study, we have used bovine fasciculata cells permeabilized in situ to identify the phosphorylated proteins and to investigate the manner in which the cytoskeleton components may act together in any subsequent reorganization of the cell. The main cytoskeletal proteins namely vimentin, tubulin, actin, and the associated protein myosin light chain were identified on polyacrylamide gel electrophoresis by their molecular weights and by Western blotting using affinity-purified monoclonal antibodies. In permeabilized cells, calcium/calmodulin promoted phosphorylation of vimentin and myosin light chain within the first 10 min. When incubation time was extended in the presence of 1 mM non-radiolabeled ATP, cell contraction was seen after 15 min. Immunofluorescent microscopy showed that actin microfilaments and myosin light chain displayed a similar pattern of distribution which indicates the actomyosin. Electron microscopy revealed the actomyosin as a dense ring around the cell beneath the plasma membrane. Intermediate filaments (10 nm) were seen within this ring which gave rise to a mixed network in which microfilaments appeared to interconnect intermediate filaments. Immunogold electron microscopy revealed that the 10-nm filaments, found within the actomyosin ring, are vimentin intermediate filaments. It is proposed that calcium/calmodulin causes phosphorylation of the myosin light chain which triggers contraction, and this process involves the intermediate filament protein vimentin. The redistribution of the cytoskeleton and hence the cell rounding is due, in part to the interconnection between vimentin intermediate filaments and actin microfilaments.(ABSTRACT TRUNCATED AT 250 WORDS)
Almahbobi
G
G
Department of Endocrinology, Prince of Wales Hospital, Randwick, NSW/Australia.
Korn
M
M
Hall
P F
PF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D007382:Intermediate Filaments / Q000187:drug effects / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D014746:Vimentin / Q000378:metabolism*; D015383:Zona Fasciculata / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D002147:Calmodulin; D014746:Vimentin; D000255:Adenosine Triphosphate; D009218:Myosins; D002118:Calcium