DYNAMICS OF THE NEURONAL CYTOSKELETON

轴突要长到很远才能到达目标组织。 轴突的发育包括沿着其长度形成间质分支以及在末端生长锥处伸长。 轴突的新区域是高度动态的，并且可以响应环境线索而快速伸长、缩回和改变它们的生长方向。微管是轴突内的基本结构元素，也充当细胞器运输的铁路。 单个微管合并成一个密集的束，横贯轴突的长度。 这种密集的纤维束对于轴突轴的稳定性很重要，但不利于伴随新生长的轴突形态的动态变化。在轴突与新生长相关的区域，微管阵列必须经历戏剧性的重组，成为一种更具可塑性的形式，使单个微管能够快速重新配置。 在准备一轮的增长，微管束局部张开，并在生长锥内或在一个网站的间隙分支形成的碎片。 短的微管然后能够移动到薄片伪足和丝状伪足，从而产生轴突生长的新区域。 在某些情况下，微管继续向前移动，而在其他情况下，微管改变方向并逆行运输。 本申请的目的是更好地表征这些微管行为中的每一种如何有助于轴突生长、收缩和分支形成，并阐明每个微管行为被编排的分子线索和机制。所有的实验都将利用高分辨率成像技术来直接可视化活皮质神经元中的微管行为。 其中一个具体的目的是试图阐明细胞骨架的变化，诱导各种外在因素和细胞内钙的变化。 我们的目标是确定这些因素，已知是相关的轴突发展，诱导局部肌动蛋白丝的变化，然后允许在微管阵列的局部变化。 另一个目标是确定负责在新生长区域中使微管张开和断裂的特定蛋白质。 最后的目的是要阐明的电机为基础的机械，运输微管顺行和逆行的增长和收缩过程中的较量。 总之，拟议的实验将提供有关调节轴突发育的分子和机制的新信息。 这些信息对于理解轴突在正常发育和损伤后再生过程中的生长将是重要的。

Axons grow over exceedingly long distances to reach their target tissues. The development of the axon involves the formation of interstitial branches along its length as well as elongation at the terminal growth cone. New regions of the axon are highly dynamic, and can rapidly elongate, retract, and change their direction of growth in response to environmental cues. Microtubules are essential architectural elements within the axon that also act as railways for organelle transport. Individual microtubules are coalesced into a dense bundle that traverses the length of the axon. This dense bundle is important for the stability of the axon shaft, but is not conducive to dynamic changes in the morphology of the axon that accompany new growth. In regions of the axon relevant to new growth, the microtubule array must undergo dramatic reorganization into a more plastic form that permits individual microtubules to be rapidly reconfigured. In preparation for a bout of growth, the microtubule bundle locally splays apart and undergoes fragmentation within the growth cone or at a site of interstitial branch formation. The short microtubules are then able to move into lamelliopodia and filopodia that give rise to new regions of axon growth. In some cases, the microtubules continue to move forward, while in other cases, the microtubules change direction and are transported retrogradely. The goals of the present grant application are to better characterize how each of these microtubule behaviors contributes to axon growth, retraction, and branch formation, and to elucidate the molecular cues and mechanisms by which each microtubule behavior is orchestrated. All of the experiments will utilize high-resolution imaging techniques to directly visualize microtubule behaviors in living cortical neurons. One of the specific aims seeks to elucidate the cytoskeletal changes that are induced by various extrinsic factors and by changes in intracellular calcium. The objective is to determine how these factors, known to be relevant to axonal development, induce local changes in actin filaments, which then permit local changes in the microtubule array. Another aim seeks to identify the specific proteins responsible for splaying and fragmenting the microtubules in regions of new growth. The final aim seeks to elucidate the motor-based machinery that transports microtubules anterogradely and retrogradely during bouts of growth and retraction. Together, the proposed experiments will provide new information on the molecules and mechanisms that regulate the development of the axon. This information will be important for understanding axon growth during both normal development and regeneration after injury.