Synapses are highly specialized intercellular contacts having specific proteins confined to the region of contact in the plasma membrane, subjacent cytoplasm, and extracellular space. How these proteins contribute to the synaptic architecture is not yet precisely determined, nor is it understood how this complex structure is assembled beginning with an undifferentiated contact between pre and postsynaptic cells. Clusters of acetylcholine receptors (AChR), formed on the ventral membranes of rat myotubes in tissue culture, are a model system for the postsynaptic membrane of neuromuscular junctions. These clusters are made up of interdigitating domains that are either rich in AChR and its associated proteins, or are attached to the substrate with concomitant links to a cytoskeleton composed of bundled actin microfilaments or polymerized clathrin. Clusters are isolated by mechanical shearing and identified by binding of fluorescent toxin to AChR. Freeze fracture and quick-freeze, deep-etch replication reveals characteristic supramolecular assemblies of the cytoskeleton in the three domains. Since shearing removes most of the cell cytoplasm, the cytoplasmic membrane surface is readily accessible for immunolabeling with antibodies absorbed to colloidal gold. Separating the replicated membrane from its supporting coverslip exposes its external surface for immunolabeling and replication. Labeling techniques will determine the relative locations of particular proteins contributing to each domain. Emphasis will be on the cytoskeleton and basal lamina proteins immobilizing AChR, and on the organization of proteins responsible for filament and substrate attachment at contact domains of myotubes and focal contacts of fibroblasts. A sequence of steps was previously proposed for cluster formation, including formation of contact domains followed by clathrin-coated domains and finally by insertion of AChR and assembly of AChR domains. Observations on clusters at various stages of development will be used to verify or disprove this scheme.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS015513-10
Application #
3396297
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1979-07-01
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
10
Fiscal Year
1990
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
Pumplin, D W; Getschman, E (2000) Synaptic proteins in rat taste bud cells: appearance in the Golgi apparatus and relationship to alpha-gustducin and the Lewis(b) and A antigens. J Comp Neurol 427:171-84
Pumplin, D W; Getschman, E; Boughter Jr, J D et al. (1999) Differential expression of carbohydrate blood-group antigens on rat taste-bud cells: relation to the functional marker alpha-gustducin. J Comp Neurol 415:230-9
De Deyne, P G; O'Neill, A; Resneck, W G et al. (1998) The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its beta5 subunit. J Cell Sci 111 ( Pt 18):2729-40
Bloch, R J; Bezakova, G; Ursitti, J A et al. (1997) A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle. Soc Gen Physiol Ser 52:177-95
Luther, P W; Samuelsson, S J; Bloch, R J et al. (1996) Cytoskeleton-membrane interactions at the postsynaptic density of Xenopus neuromuscular junctions. J Neurocytol 25:417-27
Pumplin, D W (1995) The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of beta-spectrin in filaments quantitatively resembling those of erythrocytes. J Cell Sci 108 ( Pt 9):3145-54
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
Dmytrenko, G M; Pumplin, D W; Bloch, R J (1993) Dystrophin in a membrane skeletal network: localization and comparison to other proteins. J Neurosci 13:547-58

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