Regulation of kinesin-5 during neuronal development: studies on dendrites

本研究的目的是确定脊椎动物神经元在正常发育过程中有丝分裂分子马达蛋白驱动蛋白-5的功能和调控。中枢神经系统脊椎动物神经元通常具有一个长而细的轴突和几个短而粗的锥形树突，轴突具有均匀的正端远端微管阵列和混合极性微管阵列。当轴突树突的主要目的是产生一个向外的冲动到一个潜在的遥远的目标，树突的功能是收集和巩固传入的信息。驱动蛋白-5是一种同源四聚体的运动蛋白，已知对有丝分裂纺锤体的形成是必需的，最近已被证明存在于神经元中并具有功能。也称为Eg 5或kif 11，驱动蛋白-5属于一组马达蛋白，通过影响微管相对于彼此的运输对细胞分裂至关重要。该提案假设驱动蛋白-5在终末有丝分裂后神经元中的独特作用，旨在回答关于驱动蛋白-5分布和活性调节的关键问题。在实验中驱动蛋白-5被耗尽或抑制的神经元显示出更快生长的轴突和异常长且细的树突。因此，该提议的首要假设是驱动蛋白-5作为“制动器”来调节这些神经突的生长和发育。从机制上讲，假设驱动蛋白-5通过限制微管通过其他分子马达运输到轴突和树突中来减弱轴突和树突的生长。以前的工作是在轴突上完成的，这里的重点是树突。通过三个不同的具体目标探索驱动蛋白-5的调节：第一个目标是确定驱动蛋白-5在树突状微管阵列的发育和维持中的功能;第二个目标是确定驱动蛋白-5的苏氨酸残基926磷酸化的调节作用;第三个目的是确定驱动蛋白-5是否识别特定的微管后，翻译修饰，以便在树突成熟时富集树突。这三个目标的拟议研究利用了最先进的细胞生物学分析，包括活细胞成像技术、定量免疫荧光和复杂的RNA干扰方法。分子马达通过调节微管极性模式强烈影响树突特征的假设可能对神经系统疾病的进展和潜在治疗具有深远的意义。

This proposal aims to determine function and regulation of a mitotic molecular motor protein called kinesin-5 in vertebrate neurons during their normal development. Central nervous system vertebrate neurons typically possess one long thin axon with a uniformly plus-end-distal microtubule array and several short thick tapering dendrites with mixed polarity microtubule array. While the axon¿s main purpose is to generate an outgoing impulse to a potentially faraway target, dendrites function to gather and consolidate incoming information. Kinesin-5, a homotetrameric motor protein known to be essential for the formation of the mitotic spindle, has recently been shown to be present and functional in neurons. Also called Eg5 or kif11, kinesin-5 belongs to a group of motor proteins that are crucial for cell division by influencing the transport of microtubules relative to one another. This proposal hypothesizes a unique role for kinesin-5 within the terminally post-mitotic neuron and aims to answer key questions on the regulation of kinesin-5¿s distribution and activity. A neuron where kinesin-5 is experimentally depleted or inhibited displays faster growing axons and abnormally long and thin dendrites. Therefore, the overarching hypothesis of this proposal is that kinesin-5 acts as a ¿brake¿ to modulate the growth and development of these neurites. Mechanistically, the hypothesis is that kinesin-5 attenuates the growth of axons and dendrites by limiting the transport of microtubules into them by other molecular motors. Previous work was done on axons; the emphasis here is on dendrites. Regulation of kinesin-5 is explored through three different specific aims ¿ the first aimed to determine kinesin-5 function in development and maintenance of the dendritic microtubule array; the second aimed to determine the regulatory role of kinesin-5 phosphorylation of threonine residue 926; and the third aimed to determine whether kinesin-5 recognizes specific microtubule post-translational modifications in order to become enriched in dendrites as they mature. Proposed studies in these three aims utilize cutting edge cell biological analyses including live-cell imaging techniques, quantitative immunofluorescence and sophisticated RNA interference approaches. The hypothesis that molecular motors strongly influence dendritic characteristics through regulation of microtubule polarity patterns may have profound implications for the progression and potential treatments of diseases of the nervous system.