Development of live-cell probes to investigate tubulin post-translational modifications in neuronal regeneration

项目摘要/摘要 周围神经系统(PNS)的损害可通过多种情况引起 包括创伤、代谢失衡和化疗。神经细胞损伤发生在他们长时间和 脆弱的突起(即轴突)会受到破坏，例如，伸展或切断。与三叉神经节神经元相比， 受损的中枢神经系统(CNS)神经元的再生能力极其有限，通常 导致终生残疾。损伤的周围神经可发生轴突的再生生长 然而，系统功能的恢复往往是不完整的，导致运动和感觉功能的丧失 并经常发展为神经病理性疼痛。 损伤轴突的再生被认为涉及微管动力学和后 翻译修饰(PTM)。微管是α，β-微管蛋白的动态聚合物，是微管的核心 轴突和树突的结构元素。微管也可以作为分子马达蛋白的轨迹。 (动蛋白，细胞质动力蛋白)在突触终末和神经细胞之间运输各种货物 索玛。损伤后，微管迅速碎裂，这一过程被称为沃勒式。 退化。在发育中的神经元和轴突再生过程中，神经元生长锥中的微管 轴突轴突在轴突引导和轴突延长中起着关键作用。 MT功能的调节在很大程度上是通过α-和β-微管蛋白亚型的差异表达实现的， 以及通过微管聚合物内的微管蛋白亚基的PTMS。微管亚型与PTMS的研究 天生依赖于需要细胞或组织的固定和渗透的抗体，这严重限制了 我们对微管蛋白PTMS的时空成分的理解。我们已经开发了一条管道，可以让 美国将为微管蛋白PTMS生产活细胞探针。在这里，我们将生成微管蛋白亚型和PTM的探针 与神经元退化和再生相关，并用它们来绘制 三叉神经节和中枢神经系统损伤后微管细胞骨架的变化。

PROJECT SUMMARY/ABSTRACT Damage to the peripheral nervous system (PNS) can be incurred through a wide spectrum of conditions including trauma, metabolic imbalances, and chemotherapy. Neuronal injuries occur when their long and fragile processes (i.e. axons) are damaged, for example by stretching or severing. Compared to PNS neurons, the regenerative capacity of injured central nervous system (CNS) neurons is extremely limited, typically resulting in life-long disability. Regenerative growth of axons can occur in the injured peripheral nervous system, however, restoration of function is often incomplete, resulting in loss of motor and sensory function and frequently the development of neuropathic pain. Regeneration of an injured axon is thought to involve changes in microtubule dynamics and post- translational modifications (PTMs). Microtubules are dynamic polymers of α,β-tubulin and are the core structural element of axons and dendrites. Microtubules also serve as tracks for molecular motor proteins (kinesin, cytoplasmic dynein) that transport various cargos between synaptic terminals and the neuronal cell soma. Following injury, microtubules undergo rapid fragmentation, a process known as Wallerian degeneration. In developing neurons and during axon regeneration, microtubules in the neuronal growth cone and axon shaft play key roles in axon guidance and axon elongation. Regulation of MT function is largely achieved by the differential expression of α- and β-tubulin isotypes, and by PTMs of tubulin subunits within the microtubule polymer. Studies of microtubule isotypes and PTMs inherently rely on antibodies that require fixation and permeabilization of the cell or tissue, which severely limits our understanding of the spatiotemporal component of tubulin PTMs. We have developed a pipeline that allows us to generate live-cell probes for tubulin PTMs. Here, we will generate probes for tubulin isotypes and PTMs relevant to neuronal degeneration and regeneration and use them to map the spatiotemporal patterns of changes to the microtubule cytoskeleton following injury to the PNS and CNS.