The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

Many species of the genus Vorticella are common ciliates living in many types of aquatic habitats. Their distinctive, contractile stalk anchors the unicellular body to a substrate. Contraction of the cell is calcium‐driven and independent of adenosine triphosphate (ATP), relying on the presence of calcium‐binding proteins. A coupled mechanochemical model accounts for the coiling of an elastic stalk and the binding of calcium to the calcium‐binding proteins. Stalk elongation is driven by elastic energy stored in the coiled stalk and the rate of elongation is controlled by calcium dissociation from calcium‐binding proteins and its re‐sequestration into internal stores. Recently, the taxonomic status of the subclass Peritichia has changed radically based on molecular studies. The former order Mobilida was removed from the subclass and elevated to the level of a separate subclass. Phylogenetic studies using small subunit ribosomal ribonucleic acid (ssu rRNA) and internal transcribed spacer (ITS) sequences place the Peritrichia closer to the subclass Hymenostomatia and the Mobilia closer to the subclass Peniculia. Key Concepts: Contractility in Vorticella relies on a calcium‐driven, ATP‐independent system that depends on calcium‐binding proteins. Vorticella can be used as a bio‐indicator for the evaluation of polluted waterways. Vorticella and similar filter‐feeding ciliates are used to clarify sewage in waste treatment plants. Vorticella is a useful organism for the study of exocytosis by examining production and release of granules that form the extracellular matrix of the stalk and the adhesive pad. Vorticella stalk contraction and relaxation is dependent of the presence or absence of calcium ions, this property can be exploited in a microelectromechanical system (MEMS) to perform linear work generated by spasmonemal contraction and controlled by calcium levels. Chemical analysis of the sticky material that anchors the stalk of Vorticella firmly to its substrate could result in the identification of a new type of adhesive material that sets in water.

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.

Our laboratory studies how the contractile cytoskeleton contributes to the process of phagocytosis. Because of its larger size and ease of manipulation, we chose the macrostomal cell of Tetrahymena vorax as our model for analysis of the distribution of proteins of prominent filamentous structures within the large oral apparatus (OA). Previous work in our laboratory identified centrin as a component of the fine filamentous reticulum (FFR) and actin and tetrin as colocalizing components of the coarse filamentous reticulum (CFR) and cross‐connectives (CC) (J. Eukaryot. Microbiol., 51:253–257). Our new data also show that actin coimmunoprecipitates with tetrin proteins, confirming our actin–tetrin colocalization results. Because of its positioning around the cytostome, the actin‐containing CFR/CC is a logical candidate for involvement in phagosome “pinch‐off” following prey ingestion. We have analyzed this process by employing an assay that uses the addition of calcium to induce phagosome formation. We show that inhibitors of actin are able to block this event, indicating that actin is necessary for phagosome “pinch‐off”. The OA also contains precisely arranged arrays of microtubules. We have examined the spatial relationship of the microtubular arrays to the distribution patterns of centrin, actin and tetrin, and found that tubulin and centrin fluorescences overlap in the region of the undulating membrane (UM). Tubulin fluorescence overlaps with actin and tetrin labeling at the inner edge of the CFR, where the CFR and UM converge. In addition to ciliary and oral rib labeling, tubulin antibodies also recognize the outer microtubule bundle (OMB), which delineates the right and posterior boundary of the OA.