The ability to polarize is a fundamental property of living cells, whether they are epithelial, nerve, or migrating cells, or simply growing cells that have to pick an axis for cell division. To begin to understand the structural basis for cell polarity, we employ the budding yeast because exquisite molecular and classical genetics, biochemistry and cell biology are available in this organism. Moreover, the basic principles that yeast uses to polarize its secretory pathway, and probably how it segregates its organelles during cell division, are very similar to the mechanisms employed by vertebrate cells. In the current period, we have shown that yeast has a special organizing center that drives the assembly of tropomyosin-containing actin cables. These cables are the mechanical substrates for polarized movement by the unconventional myosin-V encoded by yeast MYO2. We have shown that Myo2p binds through its tail to secretory vesicles and transports them down the actin cables for polarized growth. We have also found that the initial alignment of the nucleus with the axis of cell division is achieved through the association of the Myo2p cargo domain with Kar9p to deliver it into the bud. We propose three specific aims that build on these studies. First, we propose to identify components involved in assembling and regulating the polarized actin cables. We focus on the yeast formins, Bnilp and Bnrlp, as these appear to be key scaffolding proteins involved in the process of cable formation. Through genetic analysis we propose approaches to identify components involved in actin cable assembly, and then, based on these studies, propose approaches to reconstitute this process in vitro. Second, we propose to examine what other organdlies might be segregated by Myo2p, and approaches to identify molecules that allow Myo2p to transport specific cargoes. Third, since Myo2p is the best understood member of the myosin-V superfamily, we propose to analyze the structure of its cargo binding domain by both dissecting specific activities genetically as well as determining its structure at the atomic level. These studies will provide a foundation for understanding how yeast sets up a polarized cytoskeleton and in turn uses it to target polarized growth and segregate organelles during the cell cycle. Since homologues of molecules critical for these processes in yeast have been associated with defects in mice and in human diseases, this work should be of general relevance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-CDF-4 (02))
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Deatherage, James F
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Cornell University
Schools of Arts and Sciences
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Shin, Myungjoo; van Leeuwen, Jolanda; Boone, Charles et al. (2018) Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables. Mol Biol Cell 29:923-936
Lwin, Kyaw Myo; Li, Donghao; Bretscher, Anthony (2016) Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo. Mol Biol Cell 27:2450-62
Donovan, Kirk W; Bretscher, Anthony (2015) Head-to-tail regulation is critical for the in vivo function of myosin V. J Cell Biol 209:359-65
Donovan, Kirk W; Bretscher, Anthony (2015) Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process. J Cell Biol 210:181-9
Xu, Li; Bretscher, Anthony (2014) Rapid glucose depletion immobilizes active myosin V on stabilized actin cables. Curr Biol 24:2471-9
Wayt, Jessica; Bretscher, Anthony (2014) Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains. Mol Biol Cell 25:2817-27
Viswanatha, Raghuvir; Bretscher, Anthony; Garbett, Damien (2014) Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells. Biochem Soc Trans 42:189-94
Bretscher, Anthony (2013) Deconstructing formin-dependent actin cable assembly. Proc Natl Acad Sci U S A 110:18744-5
Chernyakov, Irina; Santiago-Tirado, Felipe; Bretscher, Anthony (2013) Active segregation of yeast mitochondria by Myo2 is essential and mediated by Mmr1 and Ypt11. Curr Biol 23:1818-24
Liu, Wenyu; Santiago-Tirado, Felipe H; Bretscher, Anthony (2012) Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation. Mol Biol Cell 23:412-22

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