Kinetochore Protein Functions in Synaptogenesis

从果蝇突变筛选胚胎神经肌肉接头(NMJ)的形成缺陷， 我们已经了解到，动粒复合体成分的突变是转化所必需的。 生长锥体形成形状正确的突触连接。这些蛋白质的丢失也会改变细胞的结构 感觉树突。在培养的哺乳动物神经元中，动粒蛋白似乎也指导着发育： 它们被RNAi击倒导致在海马树突上形成过多的丝状突起。这 是动粒的一种全新的功能，这是一种蛋白质复合体，以前只在 染色体的着丝粒，在分裂细胞时需要它来“捕捉”和稳定纺锤体微管 从而使染色体能够分离到子代细胞。神经元表型不能 可以用染色体力学中的缺陷来解释，因此我们假设 “神经着丝粒”，这一功能很可能涉及着丝粒的核心特性： 与微管的正端结合并稳定它们的能力。我们建议检验一个假设，即 与着丝粒非常相似的复合体将在有丝分裂后神经元的局部发挥作用，以帮助 动态生长锥体微管转化为稳定的突触微管束以及类似的 以稳定树突状微管，从而阻止树枝状生长。该提案利用了 果蝇和哺乳动物系统的优点。因此，本提案的目标1试图将 更深入地研究了胚胎果蝇NMJ和海马树突处缺陷的性质。《目标2》深入探讨 通过询问结构功能研究是否支持 动粒结构必须与微管结合的假说。目标3侧重于微管 细胞骨架，并询问突变体中是否存在微管动力学和稳定性缺陷，以及 动粒蛋白的这一新角色如何与我们对改变生长过程的知识相吻合 球果变成成熟的末端，并决定树枝的形态。

From a Drosophila mutant screen for defects in the formation of the embryonic neuromuscular junction (NMJ), we have learned that mutations in components of the kinetochore complex are needed for the transformation of a growth cone into a correctly shaped synaptic connection. Loss of these proteins also alters the structure of sensory dendrites. In cultured mammalian neurons, kinetochore proteins also appear to guide development: their knockdown by RNAi causes an excess of filipodia like protrusions to form on hippocampal dendrites. This is a completely novel function for the kinetochore, a protein complex previously known only to function at the centromere of chromosomes where it is required in dividing cells to “catch” and stabilize spindle microtubules and thereby enable the segregation of chromosomes to the daughter cells. The neuronal phenptypes cannot be explained by defects in chromosome mechanics and therefore we hypothesize a postmitotic function for a “neuro-kinetochore”, a function that is likely to involve the same core property of the centromeric kinetochore: the ability to bind to the plus-ends of microtubules and stabilize them. We propose to test the hypothesis that a complex very much akin to that found at centromeres will function locally in post-mitotic neurons to assist in the transformation of dynamic growth cone microtubules into stable bundles of synaptic microtubules and similarly to stabilize dendritic microtubules and thereby arrest dendrite growth. The proposal makes use of the advantages of both Drosophila and mammalian systems. Aim 1 of this proposal therefore seeks to characterize in greater depth the nature of the defects at the embryonic fly NMJ and in hippocampal dendrites. Aim 2 delves into the mechanism underlying the phenotype by asking whether structure function studies support the hypothesis of a kinetochore-like structure that must bind to microtubules. Aim 3 focuses on the microtubule cytoskeleton and asks whether there are defects in microtubule dynamics and stabilization in the mutants, and how this novel role for kinetochore proteins fits into our knowledge of the processes that transform growth cones into mature endings and determine the morphology of dendrites.