Function of an Unconventional Myosin in Tetrahymena

Cuny Brooklyn College, Brooklyn NY

Investigators

Abstract

Intellectual Merit: This project concerns in depth functional analysis of a novel unconventional myosin from the free living protozoan model organism, Tetrahymena thermophila. Myosins are a group of protein biomolecular motors that are generally associated with subcellular microfilaments made of a protein called actin; linkage of myosin molecules to various other subcellular particles or other structures results in movement of those particles or structures along the actin microfilament. Perhaps the most well-known cellular function of "conventional" myosin is that of muscle contraction, in which a linear array of myosin molecules move along actin filaments to cause shortening of entire cells. However, myosins are also involved in many other subcellular motility functions, ranging from the movement of vesicles or granules along the length of cellular extensions such as axons to the belt-like constriction of cell membrane during cell division that results in the formation of two daughter cells from the original dividing cell. In recent years, quite a few different molecular kinds (classes) of myosins have been discovered that are "unconventional" (i.e., different from the two myosin classes found in high abundance in animal tissues). In this project, the myosin under study is Myo1p, a divergent unconventional myosin from Tetrahymena that is not assigned to any of the known myosin classes. In prior NSF-funded work, Dr. Gavin disovered MYO1 and characterized the phenotype from a MYO1 knockout. Results from that work demonstrated that Myo1p is involved in two fundamental cellular processes of this protozoan: phagocytosis and nuclear motility. The predicted full-length Myo1p primary structure has a molecular mass of 210,889 Daltons and contains a region of predicted coiled coil, a 136-aa myosin tail homology 4 (MyTH4) motif, a 308-aa Band 4.1, ezrin, radixin, moesin homology (FERM) motif, a putative calmodulin-binding (IQ) motif that is located in the tail domain rather than the usual neck-domain location, and a 124-aa C-terminus. In the MYO1 knockout strain, the rate of phagosome formation was reduced, and macronuclear elongation often failed to be completed. Further studies of the knockout strain revealed that phagosomes moved randomly in the cytosol in contrast to directed movement toward the posterior end in wild-type cells. A conclusion of these studies of phagocytosis is that directed motility of phagosomes requires actin filaments and Myo1p. How phagosome-associated actin and Myo1p interact to effect directed (as opposed to random) motility of phagosomes is unknown and is the focus of this new project. The general hypothesis is that conserved and/or non-conserved regions in the tail domain of Myo1p target this myosin to its sites of action where MyTH4 and/or FERM mediates organization of actin filaments. If Myo1p is localized and linked to the site of action solely by the tail domain, over-expression of tail domain fragments that contain sufficient targeting information would replace endogenous Myo1p and thereby inhibit Myo1p function. Strains with inducible expression of tagged, tail-domain fragments that contain conserved motifs and/or nonconserved regions will be constructed. Germline gene replacements would construct different strains that express a tagged truncated Myo1p in which regions of the tail domain have been deleted. Anti-epitope tag antibodies and antibodies directed against regions of the tail domain will localize tail domain fragments. Immunostaining with antibodies directed against the Myo1p motor domain will demonstrate whether or not over-expressed tail fragments have replaced endogenous Myo1p. Double labeling with anti-Myo1p and anti-actin antibodies will localize Myo1p in relation to actin in wild-type and transformant cells. It is anticipated that Myo1p would localize either transiently or permanently to phagosomes or phagosome-associated actin and to regions near the macronucleus. Functional analyses will employ assays for phagosome motility and a morphometric analysis for identification of macronuclear aberrations in mass cultures of Tetrahymena. Immunoprecipitation and co-sedimentation assays will determine whether or not specific tail domain motifs associate with actin and induce bundling or cross-linking of actin in vitro. These studies have implications far beyond the Tetrahymena model. Internalization of particulates and fluids through phagocytosis and endocytosis is of fundamental importance to diverse cell types including protozoa, apoptotic cells, and professional phagocytes of the immune system. The underlying basis for macronuclear elongation may be related to nuclear migration and positioning that take place during development in many cell types and are known to involve cytoskeletal elements. Broader Impacts: This project will provide research training for several Brooklyn College undergraduates and would integrate a cell biology laboratory course. Outreach to a new science high school geared to educating minority urban students will involve high school students engaged in research in the PI's laboratory and workshops for high school teachers.