One of the earliest events in the formation of a synapse between nerve and muscle -- the neuromuscular junction -- is the aggregation of acetylcholine receptors (AChR) in the region of the muscle membrane contracted by nerve. Proteins of the cytoskeleton and the extracellular matrix have been implicated in this process. Little is known, however, about the organization of these proteins in the postsynaptic membrane as synaptogenesis proceeds. We will address this question by studying the formation of the neuromuscular junction in tissue culture. Cocultures of nerve and muscle cells from Xenopus laevis embryos form neuromuscular junctions that resemble the junctions of mature animals. Synaptogenesis in vitro proceeds through several distinct stages that are readily recognized when fluorescent derivatives of alpha-bungarotoxin are used to mark the postsynaptic accumulations of AChR. We will use immunofluorescence techniques: (i) to identify the extracellular and cytoskeletal proteins associated with synapses; and (ii) to determine the stage of synapse formation at which these proteins become associated with the postsynaptic region. We will use quick freeze, deep eton, rotary replication methodology at the ultrastructural level; (iii) to visualize the macromolecular arrays (e.g., microfilaments, coated membrane, extracellular matrix) that are present at the postsynaptic membrane at different stages of synaptogenesis; and (iv) to localize extracellular and cytoskeletal proteins to these synaptic structures by immunogold labeling. These studies should identify the proteins that are used to assemble the postsynaptic membrane of the neuromuscular junction, localize them to particular postsynaptic structures, and elucidate the sequence of assembly of these structures during synaptogenesis. These are essential steps if we are eventually to understand synapse formation at the molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS027171-01
Application #
3413365
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1989-06-01
Project End
1992-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Bloch, R J; Sealock, R; Pumplin, D W et al. (1994) Association of acetylcholine receptors with peripheral membrane proteins: evidence from antibody-induced coaggregation. J Membr Biol 138:13-28
Luther, P W; Samuelsson, S J; Pumplin, D W et al. (1994) Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells. Cell Motil Cytoskeleton 28:179-93
Samuelsson, S J; Luther, P W; Pumplin, D W et al. (1993) Structures linking microfilament bundles to the membrane at focal contacts. J Cell Biol 122:485-96