Gpi-Anchored Proteins In Ciliated Protozoa: From Ca++ Signaling To Mitochondrial Extrusion
Ciliates are an ancient eukaryotic lineage comprised of roughly 7,000 extant species. These include historically important models for the study of cell biology and genetics, most notably, Tetrahymena and Paramecium. One of the characteristic features of ciliates is their extensive cell surface comprised of plasma and ciliary membranes enriched in GPI-anchored proteins known as immobilization antigens or i-antigens. Antibodies against these proteins trigger a number of behavioral changes in ciliates including discharge of cortical secretory granules and arrest of cell movement (hence the term immobilization antigens). Although ciliate Immobilization antigens were discovered more than a hundred years ago, the signaling mechanisms underlying these effects are still unknown. To investigate transmembrane signaling events in response to i-antigen clustering we used Ichthyophthirius multifiliis, a parasitic ciliate, and Tetrahymena thermophila, a well-studied freeliving ciliate as model systems. Using a variety of molecular and cell biological techniques we show that antigen clustering is accompanied by mobilization of intracellular Ca++ and the formation of membrane aggregates (blebs) at the cell surface that migrate to the tips of cilia where they are shed. Remarkably, cross-linking of i-antigens also leads to mitochondrial extrusion both in Tetrahymena and Ichthyophthirius. Release of mitochondria from intact cells was shown directly by negative stain and thin-section transmission electron microscopy. Using confocal imaging in conjunction with antibodies against HSP60 and ATP synthase, extruded mitochondria were shown to co-localize with plasma membrane blebs. Mitochondrial extrusion appears to be Ca++ dependent and can be induced in response to heat shock. Cells survive the response and regain their normal architecture and swimming behavior following the extrusion. Several recent reports have suggested that mitochondria can be jettisoned from mammalian cells under conditions of oxidative stress. While the precise mechanisms responsible this phenomenon are unclear, the fact that protozoa and mammalian cells are both capable of ejecting their mitochondria would strongly suggest the process is evolutionarily conserved, and raises interesting questions regarding the advantages this phenomenon may have for cells.
dissertation or thesis