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

Genome-Wide Characterization of Tetrahymena thermophila Chromosome Breakage Sites. II. Physical and Genetic Mapping

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro- and micronuclear genetic, physical, and whole-genome sequence maps

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3.

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3.

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5

Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena

Summary: The ancestral gamete fusion protein, HAP2/GCS1, plays an essential role in fertilization in a broad range of taxa. To identify factors that may regulate HAP2/GCS1 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2/gcs1 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for membrane pore formation following mating type recognition and adherence. GFU2 is predicted to be a single-pass transmembrane protein, while GFU1, though lacking obvious transmembrane domains, has the potential to interact directly with membrane phospholipids in the cytoplasm. Like Tetrahymena HAP2/GCS1, expression of GFU1 is required in both cells of a mating pair for efficient fusion to occur. To explain these bilateral requirements, we propose a model that invokes cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in Tetrahymena’s multiple mating type system