AbstractSea urchin coelomocytes contain an actin‐based cytoskeleton that undergoes major organizational changes as the cells transform from one morphology (petaloid) to another (filopodial). The molecular mechanisms directing and regulating this cytoskeletal reorganization are not well understood; Ca2+has been implicated, but the specific targets of its action have not been identified. Since the effect of Ca2+on a variety of cellular processes has been shown to be mediated by the Ca2+‐binding protein calmodulin, we investigated the role of this protein in coelomocyte morphological transformation. The calmodulin inhibitory drugs trifluoperazine, chlorpromazine, and calmidazolium were shown to inhibit coelomocyte morphological transformation in response to hypotonic shock in a dosedependent fashion and at concentrations consistent with their reported potencies as anti‐calmodulin agents. Calmodulin isolated from coelomocytes using trifluorophenothiazine affinity chromatography co‐migrates with bovine brain calmodulin on 15 SDS‐polyacrylamide gels and demonstrates a characteristic shift in electrophoretic mobility in the presence of Ca2+. Another diagnostic for calmodulin, Ca2+‐dependent activation of exogenous 3':5' cyclic AMP phosphodiesterase, was demonstrated by whole coelomocyte homogenates, heat‐treated homogenates, and the affinity purified coelomocyte protein. Localization of calmodulin in coelomocytes by indirect immunofluorescence reveals an association of calmodulin, at least in part, with the actin‐based cytoskeleton. Calmodulin‐binding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using125I‐calmodulin overlay procedures. Ca2+‐dependent calmodulin‐binding in these preparations was demonstrated for all but the Mr30,000 and 20,000 coelomocyte polypeptides. The majority of the calmodulin‐binding proteins identified in whole petaloid coelomocyte preparations are also found in Triton X‐100 insoluble cytoskeletal fractions. Immunoblotting with antiserum raised against chicken erythrocyte alpha‐spectrin suggests that the 240,000 Mrcalmodulin‐binding polypeptide corresponds to coelomocyte alpha‐spectrin. This protein was enriched in isolated coelomocyte filopodia where, we propose, it serves an analogous function to its counterpart in erythrocytes, in linking the actin‐cytoskeleton to the plasma membrane. Thus, calmodulin is present in coelomocytes and possibly participates in the morphological transformation of these cells through regulation of cytoskeletal and/or
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