Characterization of Vorticella convallaria calcium‐binding centrin proteins

Abstract

The stalked ciliate, Vorticella convallaria, is a good model system to study mechanochemical motility because its contractile organelles (spasmoneme and myonemes) use a mode of contraction that differs from most other eukaryotic motile systems. Since calcium triggers this contraction, we have undertaken the molecular characterization of the calcium‐binding proteins associated with these organelles. We have isolated and identified seven unique centrin‐like cDNAs from V. convallaria. Each encodes an acidic protein of approximately 20‐kDa, containing a unique N‐terminus and four potential calcium‐binding domains. We predict that each centrin has a distinct function within the cell. To define these functions, we have initiated immunofluorescence localization studies utilizing various anti‐centrin antibodies. Western analysis indicates that each antibody recognizes a distinct protein or subset of proteins in Vorticella. Using these antibodies, we have localized centrin to various structures within the cell; myonemes, spasmoneme, and the oral apparatus. Because each of these antibodies recognizes a different protein on Westerern analysis, we conclude that a number of calcium‐binding proteins are associated with the contractile organelles. To further characterize this gene family, we have initiated immunolocalization at the ultrastructural level. This will permit subcellular localization of all Vorticella centrins and enable us to dissect the function of this multi‐gene family.