Analysis of centrin-, actin-, and tetrin-containing filaments in the Tetrahymena vorax macrostomal oral apparatus and their relation to the microtubule cytoskeleton

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.