Umeå University's logo

The cilium is a protrusion of cell membrane. Both the protein and lipid contents of cilia are different from those of other parts of the cell membrane. While the transport of proteins into and out of cilia has been intensively studied, much less is known about how the lipid content of ciliary membranes is regulated. TAT-6 is a P4-family ATPase that is expressed in C. elegans ciliated neurons whose endings are exposed to the environment. To study the function of TAT-6 and that other translocases in lipid transport in C. elegans ciliated neurons, I developed a technique to allow labelling of cilia with lipids. For the first time I used fusogenic liposomes to study the roles of all the TAT proteins in this organism in maintaining the lipid asymmetry in this organelle. Assessment the cilia with these liposomes showed that TAT-5 and TAT-1 translocase activities promote the transport of phosphatidylethanolamine (PE) and phosphatidylserine (PS) respectively and TAT-6 has an overlapping function in transporting both phospholipds. In C. elegans males, certain ciliated neurons release extracellular vesicles (EVs). The cilium is a site of EV biogenesis and shedding. I found that ciliated neurons in tat-6 mutant males produced significantly fewer EVs than those in wild type. tat-1, tat-5 and pad-1 mutants, however, produced far more EVs than those in wild type. PPK-3, CUP-5 and LMP-1 are proteins necessary for endolysosomal trafficking and lysosomes biogenesis, a process in which TAT-1 has previously been shown to function in C. elegans intestinal cells. I found that, like tat-1 mutants, ppk-3, lmp-1 and cup-5 mutant males release significantly greater numbers of EVs from cilia compared with wild-type. I found that increasing and decreasing the cGMP signaling cause defects in the response and turning behavior in male C. elegans respectively. Exposing wild-type males to high levels of 8-Bromoguanosine 3′,5′-cyclic monophosphate strongly reduced response behavior. Males mutant for odr-3, which encodes a G protein were defective in response. Overall my investigations indicate that the regulation of lipid asymmetry and phospholipid transport is required for proper cilia function in C. elegans, that intercellular trafficking and lipid composition have important roles in EVs biogenesis, and that different TAT proteins can affect the size and number of EVs produced. I also showed that in male animals, cGMP is one of the mediators in mating transduction signal and that a high level of cGMP inhibits mating response behavior in male C. elegans.