Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation.
Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha 5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.
Mermelstein
C S
CS
Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. mermelstein@ufrj.br
Rebello
M I L
MI
Amaral
L M
LM
Costa
M L
ML
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D048429:Cell Size / Q000502:physiology*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000032:analysis*; D004533:Egtazic Acid / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry*
D002614:Chelating Agents; D003598:Cytoskeletal Proteins; D004533:Egtazic Acid