Centrosomes and their functional equivalents in yeast, the spindle pole bodies (SPBs), are microtubule organizing centers that orchestrate mitotic spindle assembly, chromosome segregation, nuclear positioning, and numerous other aspects of cell motility and morphogenesis. To uncover how they function, we are developing biophysical tools to manipulate attachments between individual organizing centers and single microtubules. It is assumed that centrosomes and SPBs sustain considerable forces in vivo, and that these forces vary during the cell cycle. However, forces on microtubule organizing centers have never been directly measured in any organism, and the effects offeree on SPB/centrosome function are unknown. SPBs and centrosomes in vivo assemble and disassemble dynamically, and the number of microtubule attachments is regulated in correlation with cell cycle-dependent forces. This correlation suggests a form of mechanical feedback whereby SPB/centrosome-microtubule attachments are selectively stabilized when microtubules become forcefullyi.e., productivelyengaged with intracellular targets. We have recently developed several assays in which microtubules are nucleated in vitro from isolated budding yeast SPBs. By adapting these reconstituted assays for laser trapping and total internal reflection fluorescence (TIRF) microscopy, we will:
(Aim 1) measure the rupture strength and bending stiffness for individual SPB-microtubule attachments, and determine the molecular basis ofthese mechanical properties;
(Aim 2) determine whether and how SPBs are mechanically regulated by measuring how phosphorylation of specific components affects attachment strength, by comparing strengths across various cell cycle stages, and by testing whether mechanical tension affects attachment stability;
(Aim 3) quantify the forces sustained by SPB-microtubule attachments in vivo during various cell cycle stages by developing a force sensor based on fluorescence resonance energy transfer (FRET) for use in living yeast. Our efforts will also include comparative measurements using centrosomes isolated from animal sources, in order to begin identifying conserved aspects of centrosome mechanics and mechanoregulation.
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