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Pon Lab Research Interests

Role of the actin cytoskeleton in control of mitochondrial motility and inheritance in budding yeast:

Early studies of organelle inheritance focused almost exclusively on the nucleus. These studies revealed that transfer of the nucleus from mother to daughter cell is cytoskeleton-dependent and required for cell division. It is now clear that similar rules apply to the inheritance of other organelles. Our studies focus on mitochondria, essential organelles that must be produced from pre-existing mitochondria. We identified a series of cell cycle-linked motility events that lead to transfer of mitochondria from mother to daughter cell, retention of newly inherited mitochondria in the bud, and retention of remaining mitochondria in the mother cell. These motility events are dependent upon the cytoskeleton, the cell polarity machinery, and cell cycle regulatory pathways. Thus, mitochondrial inheritance, like nuclear inheritance, is an integral component of the cell division cycle.

We find that mitochondrial movement during yeast cell division occurs by a mechanism that is similar, but not identical, to that used by bacterial pathogens including Listeria monocytogenes for movement through the cytoplasm of infected cells. Shigella flexneri causes bacillary dysentery, a common disease with worldwide distribution. Listeria monocytogenes is a food borne pathogen responsible for meningitis, meningo-encephalitis, and premature pregnancy termination with a mortality rate of about 30%. Propulsion of these pathogens through the cytoplasm of infected cells occurs by Arp2/3 complex-mediated actin nucleation at a specific point along the bacterial surface. This actin nucleation stimulates a burst of actin polymerization, generating "actin comet tails" that propel bacteria from one infected host cell to the next. Mitochondrial movement, like that of the bacterial pathogens, is dependent upon the Arp2/3 complex. However, in contrast to bacterial patho-gens whose movement is randomly directed, mitochondria use actin cables (bundles of actin filaments that run along the mother-bud axis) as tracks for movement from the mother cell to the bud.

According to our current working hypothesis the "mitochore" is functionally homologous to the kinetochore and mediates cyclic binding of mitochondrial membranes and mtDNA to actin cables for movement in the presence of an applied force. The force for movement is produced by: 1) Arp2/3 complex-stimulated actin polymerization and assembly at the interface between mitochondria and actin cables, and 2) cross-linking of mitochondria-associated actin polymers in parallel to F-actin within actin cables. The net effect of these processes is movement of mitochondria towards the barbed or fast-growing end of actin cables, and linear, polarized movement of mitochondria from mother to daughter cells.

Our efforts to understand this process include molecular dissection of the proteins that 1) mediate association of mitochondria with the actin cytoskeleton and with the Arp2/3 complex, and 2) regulate mitochondrial movement and inheritance during cell division. Given the fundamental role of mitochondria in aerobic energy mobilization and biosynthesis of molecules including amino acids, fatty acids, pyrimidines, heme and steroid hormones, it is surprising the so little is known regarding the mechanisms responsible for transmission of mitochondria from mother to daughter cell during cell division. Our studies on mitochondrial movement and inheritance are designed to fill that gap.

Actin dynamics during establishment of cell polarity in budding yeast:

Actin cables are bundles of filamentous actin, which align along the mother-bud axis and resemble actin bundles in mammalian cells (e.g. the bristle and follicular nurse cell struts of Drosophila, the brush border of intestinal epithelial cells, and the stereocilium of hair cells in the vertebrate ear). Studies with tropomyosin mutants, which show temperature-dependent loss of actin cables, implicate actin cables as tracks for movement of components from mother cell to bud including secretory vesicles, mRNA, lysomes, mitochondria, and elements required for spindle alignment. Despite the fundamental role of these structures in polarized growth and yeast cell division, the events associated with actin cable polarization and maintenance are not well understood. We visualized actin cables in living yeast using fluorescent tags and time lapse fluorescence micrscopy. We find that actin cables are dynamic structures that undergo elongation and cortical movement, and achieve their polarization by assembly and extension along the mother-bud axis. Moreover, we localized sites of actin cable assembly within the incipient bud, bud and bud neck. Future studies will focus on analysis of the role of known actin binding proteins and polarity factors in actin cable assembly, movement and disassembly. These studies will extend our understanding of other fundamental actin-dependent motility events including 1) vesicle movement during secretion and neuronal transmission, 2) development and maintenance of apical modifications in lymphocytes, intestinal brush border cells, migrating neurons and other cells, 3) asymmetric distribution of organelles and mRNA during development, and 4) polarized cell growth during host invasion by pathogenic fungi (e.g. Candida albicans, Aspergillus nidulans) responsible for human infections including thrush, vaginal candidiasis, skin infections, diaper rash, and esophagitis, as well as intestinal, urinary, central nervous system, cardiac and disseminated infections.