DYNAMICS OF THE NEURONAL CYTOSKELETON

轴突要长很长的距离才能到达目标组织。轴突的发育包括沿轴突长度方向的间隙分支的形成以及末端生长锥的伸长。轴突的新区域是高度动态的，可以根据环境信号迅速延长、收缩和改变其生长方向。微管是轴突内必不可少的结构元素，也是细胞器运输的铁路。单个微管结合成密集的束，贯穿轴突的长度。这种致密的束对轴突轴的稳定性很重要，但不利于轴突形态伴随新生长的动态变化。在与新生长相关的轴突区域，微管阵列必须经历戏剧性的重组，使其成为更具可塑性的形式，从而允许单个微管快速重新配置。在为新一轮生长做准备时，微管束在生长锥内或间隙分支形成的部位局部散开并发生碎裂。然后，短微管能够进入板足和丝状足，从而产生新的轴突生长区域。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。本研究的目标是更好地描述每一种微管行为如何促进轴突生长、收缩和分支形成，并阐明每一种微管行为的分子线索和机制。所有的实验都将利用高分辨率成像技术直接可视化活皮层神经元的微管行为。其中一个特定的目的是寻求阐明由各种外在因素和细胞内钙的变化引起的细胞骨架变化。目的是确定这些已知与轴突发育相关的因素如何诱导肌动蛋白丝的局部变化，从而允许微管阵列的局部变化。另一个目标是确定在新生长区域中负责展开和分裂微管的特定蛋白质。最终目的旨在阐明在生长和收缩期间顺行和逆行运输微管的运动机制。总之，拟议的实验将提供关于调节轴突发育的分子和机制的新信息。这一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.