SUBCELLULAR DOMAINS OF MYELINATING-GLIA: CAPTURING AXONAL CONTACT.

描述（申请人提供）：胶质细胞必须在多个轴上极化。例如星形胶质细胞、放射状胶质细胞、髓磷脂形成胶质细胞或终末雪旺细胞参与多种细胞-细胞和细胞-基质相互作用（即与神经元、内皮细胞、肌肉和基层）。这种复杂的细胞结构对神经胶质功能至关重要，但在空间上也难以接近。因此，很难分离出专门用于生化研究的亚细胞区室。因此，神经胶质细胞研究的主要障碍是分子的不可接近性

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. !