FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogeneis. To investigate the role of FER-1 in membranous organelle fusion, I first examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains which commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. I found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which may arise from FER-1 by proteolysis. By both light and electron microscopy these FER-1 proteins are localized to membranous organelle membranes. Together, these results suggest that the ferlin family members may share a conserved mechanism to regulate cell-type specific membrane fusion.In Chapter III, I present additional results toward studying the function of FER-1 using several broad-based approaches. First, I present a bioinformatics analysis of FER-1 C2 domains and the preliminary results of their calcium-dependent phospholipid binding capabilities. Second, preliminary interactions found with individual FER-1 functional domains by a yeast-two hybrid screen are discussed. Lastly, I present results from a candidate-gene approach to identify additional regulators of MO fusion, the sperm-specific synaptobrevins.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/195114 |
| Date | January 2005 |
| Creators | Washington, Nicole Leanne |
| Contributors | Ward, Samuel, Fares, Johnny, Ward, Samuel, Fares, Johnny, Nagy, Lisa, Gregorio, Carol |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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