Calmodulin and caimodulin‐binding proteins in the morphological transformation of sea urchin coelomocytes

Abstract

Sea 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 3'15' 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. Calmodulinbinding polypeptides with estimated relative mobilities of 240,000, 195,000, 170,000, 70,000, 60,000, 30,000, and 20,000 daltons were identified using '251-calmodulin overlay procedures. Ca2+-dependent calmodulin-binding in these preparations was demonstrated for all but the M, 30,000 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 M, calmodulin-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 membrane-cytoskeletal interactions.

Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge

“…This clotting process likely involves both types of large phagocytes which undergo reversible lamellipodial to filopodial changes to the cytoskeleton in vitro in response to changes in the medium ( 29 – 31 , 41 , 56 ). In vivo , these morphological changes can result in cellular clots [also termed aggregates ( 56 , 59 )], which may explain the decreases in the number of large phagocytes in response to the initial injury observed here. This decrease would be consistent with their involvement in cellular clotting and encapsulating large particles by forming syncytia ( 21 , 60 , 61 ).…”

“…Damage to the body wall that breaks the rigid test or skeleton in echinoids cannot be closed as in sea stars that contract their tissues around an injury to stop the bleeding of CF. Consequently, echinoids rely on clotting processes to prevent lethal losses of CF, which is based on both cellular and protein clots (56)(57)(58). This clotting process likely involves both types of large phagocytes which undergo reversible lamellipodial to filopodial changes to the cytoskeleton in vitro in response to changes in the medium (29-31, 41, 56).…”