140 research outputs found

    Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP)

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    In the “GFP reconstitution across synaptic partners” (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same interneurons remain unlabeled. We also generated antisera that label reconstituted GFP but neither fragment in order to amplify the GRASP signal and thereby increase the sensitivity of the method

    Kinase- and rapsyn-independent activities of the muscle-specific kinase (MuSK).

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    The muscle-specific receptor tyrosine kinase (MuSK) is co-localized with nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the skeletal neuromuscular junction, and is required for all known aspects of postsynaptic differentiation. Studies in vitro have shown that Z(+)-agrin, a nerve-derived proteoglycan, activates MuSK's kinase activity to promote clustering of AChRs and MuSK itself with a cytoplasmic, receptor-associated protein, rapsyn. These studies, however, have used soluble forms of agrin, whereas agrin is cell- or matrix-attached in vivo. We show here that immobilized (particle- or cell-attached) agrin but not soluble agrin is able to aggregate MuSK in the absence of rapsyn and that this aggregation does not require MuSK's kinase activity but does require MuSK's cytoplasmic domain. Moreover, immobilized agrin can promote clustering of AChRs by a mechanism that requires MuSK and rapsyn but does not require MuSK's kinase activity. These results imply that rapsyn and signaling components activated by MuSK kinase may be dispensable for some early aspects of postsynaptic differentiation.Peer reviewe

    Viewpoints: contrasting opinions in Neural Development.

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    Nerve-independent formation of a topologically complex postsynaptic apparatus

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    As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)–rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity
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