Programmed migrations of cells during development, front-line defense against invading microbes and the development and metastasis of malignancies all depend on the capacity of certain types of cells to crawl over solid substrates. This type of cellular locomotion is complex involving interaction between the cytoplasm and the plasma membrane, the membrane and the substrate, and coordinated movement of the membrane itself. The purpose of this proposal is to study each of these related processes in a single, simplified type of crawling cell, the spermatozoon of the nematode, Caenorhabditis elegans. Monoclonal antibodies directed against membrane antigens will be used to study the membrane dynamics on crawling sperm focusing on the mechanism that propels the directed movement of membrane proteins over the cell surface, the fate of membrane components after removal from the surface, and the biochemical characteristics of cytoplasmic pools of membrane proteins that might direct their localized insertion into the plasma membrane. C. elegans sperm contain 2-nm filaments in their pseudopods but lack microfilaments and microtubules. These filaments resemble a new class of motility structures discovered in other cells. Their cellular organization will be examined by high voltage electron microscopy. A combination of immunocytochemical and biochemical techniques will be used to identify the protein that forms these filaments and the protein that links the filaments to the plasma membrane. The role of the filaments is sperm locomotion will be defined using antibodies to inhibit their function. The long term objective of this work is to understand crawling movement in molecular detail. C. elegans sperm are well-suited for this goal because they can be manipulated genetically. Eventually, it should be possible to remove individual proteins from the cell by mutation and, thereby, deduce their exact roles in locomotion.
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