The role of the extracellular matrix in establishing Schwann cell polarity

摘要 各种细胞类型建立极性以产生复杂的形态并发挥功能。雪旺细胞 (SCs)是极化的，近轴膜面向轴突，基底外侧膜面向轴突。 细胞外基质（ECM）。先前的工作确定了已知的极性蛋白，Par-3和LKB-1/Par-4， 在髓鞘形成之前定位于近轴膜，并且在SC内是及时进展所需的。 髓鞘形成和Remak束形成。虽然SC极性对于适当的周围神经至关重要， 尽管如此，在SC内建立极性的机制仍然未知。基底层，a 与基底外侧膜邻接的ECM的专门结构被认为对于调节基底外侧膜的细胞增殖是重要的。 此外，在其他上皮细胞中，胶原-IV对于极性的建立是必需的。SCS 将胶原-IV分泌到基底层中并表达整合素α 2，整合素α 2是一种胶原-IV受体， 极性的作用。因此，我推测IV型胶原通过整合素α 2信号传导启动SC极性 并且对于rSC极性是必需的，但对于髓鞘形成是必需的。我测试这种可能性分析的作用， SC分泌IV型胶原对周围神经发育的影响，决定IV型胶原是否在SC基底膜内表达。 层是必要的和足够的SC极性，并确定SC结合伴侣的胶原蛋白-IV。 初步数据表明，SC分泌的胶原-IV对于及时髓鞘形成和Remak束是重要的。 阵这些结果表型复制了通过特异性敲除LKB 1破坏极性时观察到的结果 从SC。完成后，这项研究将对整合外源性信号产生重要影响， ECM进入内部电池状态。从而调节极性，这对周围神经健康至关重要。 此外，了解极性缺陷的结果和机制可以帮助理解 周围神经病变为了进行这些研究，我将学习各种技术，包括蛋白质 生物化学，包括蛋白质印迹和免疫共沉淀，原代细胞培养技术，CRISPR- 基于基因编辑、小鼠行为测定和病毒构建/纯化/转导， 我的赞助人乔纳·陈医生的指导我将接受职业发展技能的额外培训，例如 实验设计，展示海报/演示文稿，手稿写作和手稿审查，通过 我的赞助商，UCSF神经科学项目内的课程，以及UCSF职业办公室的研讨会 和专业发展。我相信，我在加州大学旧金山分校博士期间接受的严格培训将 使我能够追求我的长期目标，成为一个终身制的轨道在学术界的首席研究员。

Abstract Various cell types establish polarity to generate complex morphology and exert function. Schwann cells (SCs) are polarized with an adaxonal membrane facing the axon and a basolateral membrane facing the extracellular matrix (ECM). Previous work established that known polarity proteins, Par-3 and LKB-1/Par-4, localize to the adaxonal membrane prior to myelination and are required within SCs for the timely progression of myelination and Remak bundle formation. Although SC polarity is critical for proper peripheral nerve development, the mechanism for establishing polarity within SCs remains unknown. The basal lamina, a specialized structure of the ECM that abuts the basolateral membrane, is believed to be important for regulating polarity; additionally, in other epithelial cells collagen-IV is necessary for the establishment of polarity. SCs secrete collagen-IV into the basal lamina and express integrin alpha2, a collagen-IV receptor also known for its role in polarity. Therefore, I hypothesize that collagen-IV initiates SC polarity by signaling through integrin alpha2 and is necessary for rSC polarity but dispensable for myelination. I test this possibility by analyzing the role of SC secreted collagen-IV on peripheral nerve development, determining whether collagen-IV within the SC basal lamina is necessary and sufficient for SC polarity, and identifying the SC binding partner for collagen-IV. Preliminary data suggests that SC-secreted collagen-IV is important for timely myelination and Remak bundle formation. These results phenocopy those observed when polarity is disrupted by knocking out LKB1 specifically from SCs. Upon completion, this study will have important implications on the integration of extrinsic signals from the ECM onto the internal cell state. Thereby regulating polarity, which is critical for peripheral nerve health. Additionally, understanding the results and mechanisms of polarity defects can inform the understanding of peripheral neuropathies. In order to perform these studies, I will learn various techniques including protein biochemistry, including Western blot and co-immunoprecipitation, primary cell culture techniques, CRISPR- based gene editing, mouse behavioral assays, and viral construction/purification/transduction under the guidance of my sponsor Dr. Jonah Chan. I will receive additional training of career development skills such as experimental design, presenting posters/presentations, manuscript writing, and manuscript reviewing through my sponsor, classes within the UCSF Neuroscience Program, and workshops through UCSF Office of Career and Professional Development. I am confident that the rigorous training I receive during my PhD at UCSF will enable me to pursue my long-term goal of becoming a tenure track principal investigator within academia.