MYOSIN-II ACTIN INTERACTION IN MDCK CELLS - ROLE IN CELL-SHAPE CHANGES IN RESPONSE TO CA2+ VARIATIONS

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+](e) less than or equal to 5 mu M) with a loss of transe pithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal st ructure in place seems necessary to preserve the architecture of the t ight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+](e). Recent work provide d evidence of actin-myosin interactions at the filament ring, thus sug gesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and cau ses an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10 min of removal of [Ca 2+](e); recovery began immediately after replacing calcium, although i t took longer for completion. Conventional and confocal epifluorescenc e studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electro n-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction when Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+](e) variations were inhibited by cytochalasin-D (an actin-filam ent disrupting drug), by okadaic acid (an inhibitor of myosin light-ch ain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cel ls previously depleted of metabolic energy by 2,4-dinitrophenol and 2- deoxy-D-glucose preincubation. The actin-myosin mediated reversible st ructural transformation of MDCK cells in response to [Ca2+](e) poses n ew questions for the interpretation of in vitro experiments, as well a s for the understanding of epithelial function. (C) Chapman & Hall Ltd .