SUBCELLULAR DOMAINS OF MYELINATING-GLIA: CAPTURING AXONAL CONTACT.

髓鞘化神经胶质细胞的亚细胞域：捕获轴突接触。

• 批准号: 8550537
• 负责人: M. Laura Feltri
• 金额: $ 22.56万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-28 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8550537
• 关键词: Address; Afferent Neurons; Astrocytes; Axon; Basal lamina; Biochemical; Blood - brain barrier anatomy; Brain; Cell Communication; Cell membrane; Cell physiology; Cells; Complex; Cues; Endothelial Cells; Endothelium; Event; Extracellular Matrix; Fibronectins; Goals; Intercellular Junctions; Knowledge; Laminin; Membrane; Methods; Molecular; Molecular Profiling; Muscle; Myelin; Neuroglia; Neurons; Oligodendroglia; Peripheral Nerves; Physiological; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Pseudopodia; RNA; Radial; Rattus; Resources; Role; Schwann Cells; Side; Signal Transduction; Site; Stimulus; System; Western Blotting; design; innovation; migration; mutant; myelination; nervous system disorder; neuronal cell body; novel; polarized cell; response; synaptic function; tool

DESCRIPTION (provided by applicant): Glial cells must polarize on multiple axes. For example astrocytes, radial glia, myelin-forming glia or terminal Schwann cells engage in multiple cell-cell and cell-matrix interactions (i.e., with neurons, endothelial cells, muscle and basal lamina). This complex cytoarchitecture is crucial for glial function, but is also spatially inaccessible. As a result it is difficult to isolate specialized subcellular compartments for biochemical studies. Thus a major obstacle to the study of glia is the inaccessibility of molecular events occurring in relevant subcellular compartments. We have adapted a system, normally used to isolate polarized cell protrusion formed in response to soluble stimuli, to neuronal-Schwann cell interactions. We introduced the innovation of using neuronal cell membranes as stimulus, instead of soluble or extracellular matrix molecules, to mimic cell-cell interactions in glial cells. To this end we placed Schwann cells on a modified Boyden chamber with microporous filters, and exposed them to neuronal cell membranes in the bottom chamber. This causes Schwann cells polarization and extension of lamellipodia-like pseudopodia. Pseudopodia and cell bodies can then be physically separated and their contents compared. We performed proteomic and western blot analysis on these pseudopods, and found known molecules located at sites of axo-glial interactions, validating the system. We now propose to use this system to identify novel players in axo-glial interactions, using large sensory neurons and after addition of a second polarizing cue (extracellular matrix) to the Schwann cells. Next we will ask if the system can be used to study interactions between neurons and other glia, namely oligodendrocytes and astrocytes. The system can be adapted to multiple wild-type or mutant glia-cell interactions, to probe their role on protein or RNA polarization, formation of specific molecular complex or protein modification. These phenomena are relevant to physiological and pathological glial cell functions. This transformative resource could overcome the difficulty to study important glial undertakings at specialized cell junctions. !

描述（由申请人提供）：神经胶质细胞必须在多个轴上伸展。例如，星形胶质细胞、放射状神经胶质细胞、髓鞘形成神经胶质细胞或终末施旺细胞参与多个细胞-细胞和细胞-基质相互作用（即，具有神经元、内皮细胞、肌肉和基底层）。这种复杂的细胞结构对于神经胶质功能至关重要，但在空间上也难以接近。因此，很难分离专门的亚细胞区室进行生化研究。因此，神经胶质细胞研究的一个主要障碍是分子生物学的不可及性。 在相关亚细胞区室中发生的事件。 我们已经适应了一个系统，通常用于隔离极化细胞突起形成响应可溶性刺激，神经元雪旺细胞的相互作用。我们介绍了使用神经元细胞膜作为刺激，而不是可溶性或细胞外基质分子，以模拟神经胶质细胞中的细胞-细胞相互作用的创新。为此，我们将施万细胞置于带有微孔过滤器的改良Boyden室中，并将其暴露于底部室中的神经元细胞膜。这导致雪旺氏细胞极化和片状伪足样伪足的延伸。伪足和细胞体然后可以物理分离并比较它们的内容物。我们对这些伪足进行了蛋白质组学和蛋白质印迹分析，发现了位于轴胶质相互作用位点的已知分子，验证了该系统。 我们现在建议使用这个系统来识别新的球员在轴-胶质细胞的相互作用，使用大型感觉神经元和后添加第二极化线索（细胞外基质）的雪旺细胞。接下来，我们将询问该系统是否可用于研究神经元和其他胶质细胞（即少突胶质细胞和星形胶质细胞）之间的相互作用。该系统可适用于多种野生型或突变型胶质细胞相互作用，以探测它们在蛋白质或RNA极化、形成特异性分子复合物或蛋白质修饰中的作用。这些现象与神经胶质细胞的生理和病理功能有关。这种变革性的资源可以克服在专门的细胞连接处研究重要的神经胶质细胞的困难。!