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

基本信息

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
项目类别：
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

项目摘要

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极性以产生复杂的形态并发挥功能。施万细胞（SC）通过面向轴突的前轴膜和面向轴突的基底外侧膜进行极化细胞外基质（ECM）。先前的工作证实已知的极性蛋白 Par-3 和 LKB-1/Par-4，在髓鞘形成之前定位于轴突膜，并且是 SC 内及时进展所必需的髓鞘形成和 Remak 束形成。尽管 SC 极性对于正常的周围神经至关重要然而，在 SC 内建立极性的机制仍然未知。基底层，紧邻基底外侧膜的 ECM 的特殊结构被认为对于调节非常重要极性;此外，在其他上皮细胞中，IV 型胶原对于极性的建立是必需的。 SC 将 IV 型胶原蛋白分泌到基底层并表达整合素 α2，这是一种 IV 型胶原蛋白受体，也因其极性中的作用。因此，我假设 IV 型胶原蛋白通过整合素 α2 发出信号来启动 SC 极性对于 rSC 极性是必要的，但对于髓鞘形成是可有可无的。我通过分析以下角色来测试这种可能性 SC 分泌 IV 型胶原对周围神经发育有影响，决定 SC 基底细胞内是否存在 IV 型胶原层对于 SC 极性以及识别 IV 型胶原的 SC 结合伴侣是必要且充分的。初步数据表明 SC 分泌的 IV 型胶原蛋白对于及时髓鞘形成和 Remak 束很重要形成。这些结果与通过特异性敲除 LKB1 破坏极性时观察到的结果进行表型复制来自 SC。完成后，这项研究将对外部信号的整合产生重要影响。 ECM 到内部细胞状态。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帮助我们理解周围神经病。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、小鼠行为分析和病毒构建/纯化/转导我的赞助商 Jonah Chan 博士的指导。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、手稿写作和手稿审查我的赞助商、加州大学旧金山分校神经科学项目中的课程以及加州大学旧金山分校职业办公室举办的研讨会和专业发展。我相信我在加州大学旧金山分校攻读博士学位期间接受的严格培训将使我能够实现成为学术界终身教授首席研究员的长期目标。