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Mechanisms of cortical capture of microtubules specifying correct spindle polarity in S. cerevisiae

Reference	BB/E006272/1
Principal Investigator / Supervisor	Dr Marisa Segal
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Genetics
Funding type	Research
Value (£)	325,086
Status	Completed
Type	Research Grant
Start date	01/05/2007
End date	30/04/2010
Duration	36 months

Abstract

Mitotic spindle orientation is critical in asymmetric cell divisions and the generation of cell diversity. The yeast S. cerevisiae is a unique model to address how spindle orientation is achieved in a cell dividing asymmetrically. Spindle position is controlled by a programme of microtubule (MT)-cortex interactions partly set by the polarity determinant Bud6p/Aip3p. These interactions direct one spindle pole to the bud and confine the other to the mother cell. Bud6p has been characterised as a partner of yeast formins in the organisation of a polarised actin cytoskeleton. Thus, Bud6p function in spindle orientation has been traditionally linked to Kar9p, a protein guiding MTs along actin cables. Studies in our laboratory, however, have demonstrated a separate role for Bud6p in cortical capture of MTs. Bud6p typically couples shrinkage of MTs at the cell cortex with spindle pole movement even in the absence of Kar9p. Here we propose to dissect the precise mechanism for MT dynamics coupled to cortical capture at Bud6p sites and establish whether actin plays any role in this process. First, we will outline Bud6p functional domains involved in MT capture to aid in the identification of potential interactors. Second, we will apply mutant analysis and cell biology tools to explore the precise contribution of formins and actin to cortical capture in vivo. Third, we will combine genetic, proteomics and cell biology tools to characterise partners for Bud6p in cortical capture. Control of spindle orientation involves components highly conserved throughout evolution. Model systems have been invaluable for dissecting regulation of embryonic cell patterning, neurogenesis, mechanisms of centrosomal inheritance in stem cells and defining the basis for genetic disorders such as lissencephaly. Thus, our studies should provide novel molecular links for understanding how polarity factors direct cell fate determination in normal human development and their importance in disease.

Summary

Every multicellular organism consists of a variety of cell types. This diversity arises in part from asymmetric cell divisions in which cell fate determinants are segregated unequally between the two sister cells. Accordingly, they differ in their intended fate and will perform different tasks. Of particular importance is the asymmetric nature of stem-cell divisions: one daughter cell commits to differentiate, while the other remains undifferentiated, thus preserving its stem-cell potential to generate diverse cell types. We study the mechanisms underlying differential fate in the budding yeast S. cerevisiae (baker's yeast), a unicellular organism dividing asymmetrically into a larger mother cell and a bud. In yeast, asymmetric segregation (mother vs. bud-bound) of the spindle poles effectively orients the mitotic spindle, a cellular machinery that drives the accurate distribution of the genetic material. This process is partly directed by Bud6, a protein marking the bud cell inner surface to attract interactions from the spindle pole destined to the bud by a process known as cortical capture. Bud6 has the remarkable property of accumulating at two distinct locations in a temporally regulated manner: first to the surface of the emerging bud and later to the bud neck, a constriction at the boundary between the bud and the mother cell that ultimately constitutes the site for cell division. The spindle poles can respond to these changes in the intracellular landscape and become positioned within the right compartment -one pole is directed to the bud while the remaining pole is retained within the mother cell. Here we propose to identify cellular components working along with Bud6 to elucidate the mechanism for these asymmetric interactions and the basis for cortical capture. The project will entail the following steps that take advantage of the ease of genetic manipulation possible in budding yeast. First, we will exploit the use of mutations to further dissect the function of Bud6 by defining a minimal part of this protein sufficient for cortical capture. Second, we will employ mutations to disable cellular components suspected of working together with Bud6. The impact of these manipulations on processes normally driven by Bud6 will be assessed by visualising the resulting behaviour in the mutant cells by microscopy methods. Third, we will determine if these components truly interact with Bud6. We will purify Bud6 from cells under conditions that may allow the isolation of a complex containing Bud6 bound to other proteins. The composition of these complexes can be determined by established methods and tools available as a result of the elucidation of the sequence of the yeast DNA genome. Studies in model systems like yeast have proven invaluable in understanding principles of embryonic development and are likely to advance our knowledge of fundamental aspects of stem cell biology with concomitant impact in human disease and therapeutics.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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