Molecular Mechanics of the Yeast Centrosome

酵母中的中心体和它们的功能等价物纺锤体极体(SPBS)是微管 组织中心协调有丝分裂纺锤体组装，染色体分离，核定位， 以及细胞运动和形态发生的许多其他方面。为了揭示它们是如何运作的，我们正在 开发生物物理工具来操纵单个组织中心和单个组织中心之间的连接 微管。假设中心体和SPBS在体内维持相当大的力量，并且这些 在细胞周期中，力是不同的。然而，微管组织中心的力量从来没有直接 在任何生物体中都有测量，而且供体对SPB/中心体功能的影响尚不清楚。SPBS和 体内中心体动态组装和拆解，附着微管的数量为 与细胞周期依赖的力量相关地调节。这种关联表明了一种形式的机械 反馈，当微管形成时，SPB/中心体-微管附件被选择性地稳定 成为强有力的--即富有成效的--与细胞内目标打交道。我们最近开发了几个 从分离的芽生酵母SPBS中分离的微管在体外成核的分析。通过调整这些功能 重建的激光捕获和全内反射荧光(TIRF)显微镜分析方法，我们将： (目标1)测量单个SPB-微管附着体的断裂强度和弯曲刚度，以及 确定这些机械性能的分子基础；(目标2)确定SPBS是否以及如何 通过测量特定成分的磷酸化如何影响附着强度来进行机械调节， 通过比较不同细胞周期阶段的强度，以及测试机械张力是否会影响 附着稳定性；(目标3)量化SPB-微管附着在活体中所承受的力 基于荧光共振能量传递的力传感器的研制 (FRET)用于活酵母。我们的努力还将包括使用着丝粒进行比较测量 从动物来源分离出来，以便开始识别中心体机制的保守方面 机械调节。

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 forcefully—i.e., productively—engaged 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.