The role of the extracellular matrix in establishing Schwann cell polarity

细胞外基质在建立雪旺细胞极性中的作用

• 批准号: 10604797
• 负责人: Monique Lillis
• 金额: $ 4.32万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604797
• 关键词: Academia; Address; Adult; Age; Ascorbic Acid; Axon; Basal lamina; Behavioral Assay; Binding; Binding Sites; Caliber; Cell Communication; Cell Culture Techniques; Cell Maintenance; Cell Maturation; Cell Polarity; Cell physiology; Cell secretion; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Collagen Receptors; Collagen Type IV; Complex; Cues; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Defect; Demyelinating Diseases; Development; Developmental Delay Disorders; Doctor of Philosophy; Educational workshop; Electron Microscopy; Environment; Epithelial Cells; Exons; Experimental Designs; Extracellular Matrix; Extracellular Space; Extracellular Structure; G-Protein-Coupled Receptors; Genes; Goals; Health; Immunofluorescence Immunologic; In Vitro; Integrin alpha2; Integrins; Knock-out; Laboratories; Learning; LoxP-flanked allele; Manuscripts; Mediating; Membrane; Morphology; Motor; Mus; Mutation; Neuroglia; Neurosciences; PAWR gene; Perception; Peripheral Nerves; Peripheral Nervous System; Peripheral Nervous System Diseases; Phenocopy; Phenotype; Primary Cell Cultures; Principal Investigator; Process; Protein Biochemistry; Proteins; Publishing; Research; Role; STK11 gene; Schwann Cells; Sensory; Signal Transduction; Specific qualifier value; Techniques; Testing; Training; Viral; Western Blotting; Work; Writing; basolateral membrane; career; career development; cell type; experimental study; in vivo; insight; intercalation; loss of function; molecular sequence database; mutant; myelination; novel; painful neuropathy; polarized cell; posters; prevent; programs; receptor; recruit; sciatic nerve; skill acquisition; tenure track

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.

抽象的 各种细胞类型建立了极性，以产生复杂的形态和施加功能。 Schwann细胞 （SC）用轴突面向轴突的辅助膜和基底外侧膜面向两极分化 细胞外基质（ECM）。以前的工作确定了已知的极性蛋白，Par-3和Lkb-1/par-4， 在髓鞘化之前定位于辅助膜，并需要在SC中进行及时进展 髓鞘形成和Remak束组。尽管SC极性对于适当的外围神经至关重要 开发，在SC中建立极性的机制仍然未知。基底薄片，一个 邻接基底外侧膜的ECM的专业结构被认为对调节很重要 极性;另外，在其他上皮细胞中，胶原蛋白IV对于建立极性是必需的。 SCS 分泌胶原蛋白IV到基底层和表达整合素α2，这是一种胶原蛋白IV受体 在极性中的作用。因此，我假设胶原蛋白IV通过通过整合素alpha2发出信号来启动SC极性 对于RSC极性是必需的，但对于髓鞘起见是必不可少的。我通过分析的作用来测试这种可能性 SC分泌的胶原蛋白IV在周围神经发育中，确定SC基础内是否胶原蛋白 薄片对于SC极性是必要的，并且足以确定胶原蛋白IV的SC结合伴侣。 初步数据表明，SC分泌的胶原蛋白IV对于及时的髓鞘形成和Remak Bundle很重要 形成。这些结果的表观副本是通过特异性击倒LKB1时观察到的那些结果 来自SCS。完成后，这项研究将对来自 ECM进入内部细胞状态。从而调节极性，这对于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告知人们对 周围神经病。为了进行这些研究，我将学习包括蛋白质在内的各种技术 生物化学，包括蛋白质印迹和共免疫沉淀，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 我的赞助商Jonah Chan博士的指导。我将获得有关职业发展技能的其他培训，例如 实验设计，介绍海报/演示文稿，手稿写作和手稿审查 我的赞助商，UCSF神经科学计划中的课程，以及UCSF职业办公室的讲习班 和专业发展。我相信我在UCSF博士学位期间接受的严格培训将 使我能够追求成为学术界统治轨道首席研究员的长期目标。