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<p>In the central nervous system, billions of neurons interconnect with precision
to form morphologically complex and functionally diverse neural circuits. The stereotypical
fashion by which neurons assemble suggests that cell-surface molecular cues can act
as identity tags during development. These cell surface receptors allow neurons to
distinguish between circuit partners, incorrect connections and homotypic neighbors.
Multiple EGF-like domains 10 (Megf10) was previously identified to mediate homotypic
recognition of certain retinal cell types. Genetic evidence suggests that MEGF10 acts
as both ligand and receptor to initiate cell-cell repulsion. Although its significance
in cell-cell recognition has been demonstrated, the exact mechanism of how MEGF10
mediates mosaic formation remains unclear. Specifically, the biochemical basis of
MEGF10-MEGF10 interaction is largely unknown nor do we have knowledge on what molecules
are involved in signaling transduction. Further, MEGF10 is also expressed in glia
cells, but it has not been tested if this MEGF10 recognition event is neuron specific.To
address these questions, we decided to first characterize the molecular components
of the MEGF10 complex. We determined MEGF10 complex composition through co-immunoprecipitation
(co-IP) and chemical crosslinking and discovered that MEGF10 forms a lateral complex.
Truncation and co-IP studies reveal that the interacting motifs are located on the
ectodomain of MEGF10. Such binding is not restricted to MEGF10 as we also discovered
hetero-multimers between MEGF10 and MEGF12. Next, to identify other molecules in
the MEGF10 signaling pathway, we performed IP to isolate native MEGF10 interacting
complexes and conducted proteomic analysis. We found previously known MEGF10-interacting
molecules such as Dynamin1 and Traf4, as well as novel MEGF10 associating candidates.
Our identification of interacting molecules that facilitate cytoskeletal and membrane
rearrangement suggests that MEGF10 activates these cellular processes. Finally, we
characterized Müller glia organization through a genetic-based labeling method. With
a MEGF10 mutant mouse, we determined that MEGF10 is not necessary for glial array
formation. We conclude that MEGF10 has distinct functions in neurons vs. glia. This
study sets the stage to describe the molecular mechanism by which MEGF10 mediates
cell-cell recognition, potentially uncovered novel MEGF10 interactions, and distinguishes
MEGF10 neuronal function from its role in glia.</p>
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