John S. Chant
Molecular Mechanisms Controlling Cell Polarity
Our chief interests are the molecular mechanisms by which cells regulate their polarization in response to internal programs or external cues. The two experimental systems that we study are budding yeast during vegetative growth and helper T cells during communication with antigen-presenting cells. In yeast, an axis of cytoskeletal polarization is oriented under the control of an internal cellular program, while, in T cells, an axis of cytoskeletal polarization is oriented in response to an external cue, the point of contact with an antigen-presenting cell. In both systems, our goal is to understand the molecular mechanisms by which spatial cues are read and transduced to produce cytoskeletal polarization.
During yeast cell division by budding, the position of buds and associated axes of cell polarity are programmed internally by cell type: a and a cells bud in an axial pattern, whereas a/a cells bud in a bipolar pattern (see diagram below). Our most recent progress has centered around Bud10, a receptor-like transmembrane molecule involved in marking the mother-bud junction in one cell cycle for axial budding in the subsequent cell cycle. Previously, we have shown that, in the axial pattern, Bud3 protein assembles in a double ring encircling the mother-bud neck around the time of onset of mitosis. Bud3 probably assembles on the 10-nm filaments, a cytoskeletal structure which encircles the neck. The assembly of Bud3 and associated factors in the neck region produces a spatial memory in one cell cycle to guide axial polarization of the cytoskeleton, bud formation and cell division in the next cell cycle. Bud10 is an additional component of the spatial memory which marks the neck region of the mother-bud pair. Interestingly, Bud10 localization to the neck is partially dependent upon Bud3. It appears that Bud10 can localize diffusely to the mother- bud neck in the absence of Bud3, however tight localization of Bud10 in double rings requires the function of Bud3. We hypothesize (and plan to test) that Bud10 localizes crudely to the neck region by binding cell wall through its extracellular domain and that its localization is refined by binding Bud3 and associated factors. In addition, we are investigating the mechanism by which Bud10 signals to establish cytoskeletal polarization in axial orientations. Like many receptor type molecules, Bud10 possesses no obvious catalytic motif in its intracellular domain. Perhaps, clustering of Bud10 in association with Bud3 promotes docking of downstream factors such as regulators of the GTPase modules (Bud1, Cdc42) involved in bud-site selection.
Currently, we are expanding our studies of T cell polarization. Previously, we have shown that Cdc42 GTPase is important for orienting both the actin and microtubule cytoskeletons of T cells towards antigen-presenting cells. Our current focus is to determine the subcellular localization of Cdc42 and related GTPases (Rho, Rac) within the polarizing T cell and to examine the contribution, if any, of these related GTPases to T cell polarization. Finally, we are attempting to dissect the pathways downstream of Cdc42 which contribute to polarization of the cytoskeleton.