Tracking in vivo mobility of polarity determinants in yeast Asymmetric cell divisions are central to stem cell biology and the generation of cell diversity. Budding yeast is one of the best paradigms for cells dividing asymmetrically. In yeast, the polarity determinant Bud6 follows a distinctive pattern of cortical localization along the cell cycle. This pattern supports the program for astral microtubule capture instructing mitotic spindle orientation. First, Bud6 is recruited at the incipient bud. Then, Bud6 partitions between the growing bud and the bud neck cortex. Finally, Bud6 abandons the bud and accumulates at the bud neck giving rise to a ring that splits at cytokinesis. Meanwhile Num1, the anchor for dynein, occupies the opposite cortical domain (the mother cell) until anaphase when Num1 foci begin to localize to the large bud. Bud6 and Num1 may act antagonistically to demark their respective domain boundaries (in time and space). This interplay for establishing a polarised cortical landscape represents a powerful model to explore the instructive role of the cell cortex in asymmetric cell divisions. Tandem fluorescent protein timers (TFt) can be used to track protein mobility between subcellular compartments in living cells (Khmelinskii et al. 2012). Here we propose to implement this approach to follow Bud6 and Num1 dynamic partitioning and explore the mechanism for antagonistic interplay. The project involves yeast molecular genetics, live imaging and quantitative analysis of TFts. This setup will be exploited to further address trafficking and the underlying role of the cytoskeleton in Bud6 and Num1 mobility.
- Khmelinskii et al. 2012. Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat Biotechnol 30: 708-14
- ten Hoopen et al. 2012. Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae. Curr Biol. 22: 1075-83.