Function of an Unconventional Myosin in Tetrahymena

Cuny Brooklyn College, Brooklyn NY

Investigators

Abstract

Myo1p is a novel, unconventional myosin discovered by Dr. Gavin and colleagues in the model protozoan Tetrahymena thermophila that has been implicated as playing a role in two fundamental cellular processes: endocytosis/phagocytosis and nuclear motility. In a recently completed project, the Gavin laboratory used targeted gene disruption to create a Tetrahymena strain that lacked a functional MYO1 gene. In transformed MYO1 cells, food vacuoles (endosomes) formed at a slower rate than in wild-type cells, and the macronucleus frequently failed to elongate properly during amitotic cell division, resulting in grossly unequal segregation of DNA to progeny. In this renewal project, experiments will be performed to directly and specifically link Myo1p to the machinery for both endocytosis and macronuclear elongation. Two general hypotheses for Myo1p function in Tetrahymena will be tested. (1) Hypothesis: MYO1 is required for both endosome maturation and endosome recycling. The rate of endosome formation could be regulated, in part, by the rate of endosome maturation in the cytosol and subsequent endosome recycling at the membrane structure known as the cytoproct. If Myo1p is involved in endosome maturation, a MYO1-knockout strain might exhibit defects in expansion and/or acidification of nascent endosomes. If Myo1p is involved in endosome recycling at the cytoproct, a MYO1-knockout strain might have a reduced rate of endosome degradation. (2) Hypothesis: Macronuclear elongation is mediated by cooperative action of microtubules, actin microfilaments, and Myo1p. One can envision at least two, non-mutually exclusive, arrangements for the putative molecular machinery that could power macronuclear elongation. The elongation machinery could be concentrated near the midzone of the macronuclear interior or membrane. Alternatively, the machinery for elongation could be concentrated throughout the periphery of the macronucleus either directly on the macronuclear membrane or in the surrounding cytosol. In order to test these hypotheses, a full-length MYO1 sequence will have to be acquired and new reagents developed. Therefore, Project I will involve completion of MYO1 sequencing, generation of polyclonal antibodies against Tetrahymena actin and Myo1p, and creation of GFP- expression constructs for three Tetrahymena proteins: actin, tubulin, and Myo1p. Project II focuses on Myo1p function in endocytosis. Three areas of investigation will be pursued: (1) localization of actin and Myo1p to components of the endocytic pathway, (2) maturation of endosomes, and (3) endosome recycling at the cytoproct. Cells expressing either GFP-actin or GFP-Myo1p and MYO1-knockout cells expressing GFP-actin will be investigated with time-lapse confocal microscopy. Fluorescent polystyrene beads and membrane marker dyes will be used in assays for endosome maturation and endosome recycling. In project III, confocal microscopy of cells expressing either GFP-actin, GFP-tubulin, or GFP-Myo1p and MYO1-knockout cells expressing either GFP-actin or GFP-tubulin will determine whether actin microfilaments, microtubules, and Myo1p are components of a molecular machinery that could power macronuclear elongation. An in vitro motility assay will obtain direct evidence for Myo1p-powered movement of macronuclei. Functional studies of Myo1p have the potential for expanding our understanding of endocytosis /phagocytosis and nuclear motility and may reveal novel mechanisms for the role of unconventional myosins in these processes. Studies of endocytosis/phagocytosis have implications far beyond the Tetrahymena model. Cell regulation of surface receptors is mediated by endocytosis, and the phagocytic process is important for the feeding of many protists, and in metazoa it is an essential part of the immune system. The underlying basis for macronuclear motility may be related to nuclear migration and positioning that take place during development in many cell types and are known to involve cytoskeletal elements.