Personnel
Robert Burgess
Associate Staff Scientist
The Jackson Laboratory
(207) 288-6706
robert.burgess@jax.org
http://www.jax.org/staff/robert_burgess.html
Lab members: Peter Gurest, Steven Rauch, Bing Wang
Research:
Localization and distribution of agrin in the synaptic membrane
Chemical synapses are specialized sites of cell adhesion and intercellular signaling that require the precise localization of both pre- and postsynaptic proteins to maintain proper function. Agrin is an extracellular matrix molecule that is essential for the proper organization and maintenance of the neuromuscular junction. It is proposed to serve an analogous role in the central nervous system, where it localizes to a subset of synaptic types. To better understand the role of agrin in both neuromuscular junction and central nervous system synapse formation, it is necessary to determine its exact localization within the synapse, and to determine whether the presynaptic cell, postsynaptic cell, or both are responsible for its production. We will be able to address these questions using transgenic mice that express full-length agrin tagged with cyan fluorescent protein. These transgenic mice have been characterized, they produce functional agrin and the transgenic expression may be eliminated by the expression of the site-specific recombinase cre.
At the neuromuscular junction, the sites of neurotransmitter release on the motor nerve terminal are aligned directly across from invaginations in the muscle fiber that contain the highest concentrations of acetylcholine receptors. Specializations in the protein composition of the extracellular matrix determine this alignment. Agrin is made by both motor neurons and muscle fibers, but these cell types generate different isoforms of the protein that have very different activities in synapse formation. By combining the agrin-CFP transgenic mice and tissue specific cre expression, it will be possible to independently visualize muscle-derived agrin and nerve-derived agrin. If neuronally-derived agrin has a specific localization within the synapse, that would suggest that it is also contributing to the alignment of the synaptic machinery. These subsynaptic domains are separated by a few hundred nanometers, and the synaptic cleft itself is only 30-50 nanometers, so the highest microscopic resolution possible will be required to resolve these questions.
In the central nervous system, the predominant agrin isoform is a type 2 transmembrane protein, which may function in cell adhesion, similar to cadherins or neurexin/neuroligin complexes. Better visualization of agrin at central nervous system synapses would help to clarify its role in synaptogenesis. Using primary neuronal cultures from the agrin-CFP transgenic mice, it will be possible to visualize agrin on neurons and to watch its localization and redistribution on the cell during synapse formation in vitro. Since neuron/neuron synapses are much less than a micron in size, high-resolution microscopy will again be necessary to accurately assess agrin's localization within the synapse and in comparison to other synaptic proteins.
In addition to the high resolution imaging studies described above, we are also examining the role of agrin in cell adhesion and signaling using patterned arrays as cell culture substrates. This work is in collaboration with Dr. Joachim Spatz of the University of Heidelberg. The effect of agrin on neurons in vitro has been previously characterized using soluble agrin applied in the media. The focal presentation of agrin on a nano- or micro-arrayed surface will potentially have a much greater impact on establishing specialized sites of adhesion in the cell, and should more closely model how agrin is presented at sites of cell adhesion in vivo. It is our long-term goal to combine these patterned substrates with primary cells isolated from different knockout and transgenic mouse strains to dissect the molecular cues needed to establish interneuronal synapses.
The combination of high-resolution imaging studies to determine precise molecular localizations in vivo and high-precision presentation of signaling molecules to cells in vitro provides a powerful resource for the study of cell adhesion and synapse formation.
Recent Publications:
Stacy RC, Demas J, Burgess RW, Sanes JR, Wong ROL. 2005. Disruption and recovery of patterned retinal activity in the absence of acetylcholine. J Neurosci 25:9347-9357.
Patton BL, Burgess RW. 2005. Synaptogenesis. In: Developmental Neurobiology. Rao MS, Jacobson M (eds). Kluwer Academic/Plenum Publishers, New York.
Burgess RW, Peterson KA, Johnson MJ, Roix JJ, Welsh IC, O’Brien TP. 2004. Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice. Mol Cell Biol 24:1096-1105.
Buffelli M, Burgess RW, Feng G, Lobe C, Lichtman JW, Sanes JR. 2003. Genetic evidence that relative synaptic activity biases the outcome of synaptic competition. Nature 424:430-434.
Misgeld T, Burgess RW, Lewis RM, Cunningham JM, Lichtman JW, Sanes JR. 2002. Roles of neurotransmitter in synapse formation: Development of neuromuscular junctions lacking choline acetyltransferase. Neuron 36:271-284.
Burgess RW, Dickman DK, Nunez L, Glass DJ, Sanes JR. 2002. Mapping sites responsible for interactions of agrin with neurons. J Neurochem 83:271-284.
Bhattacharya S, Stewart BA, Niemeyer BA, Burgess RW, McCabe BD, Lin P, Boulianne G, O’Kane CJ, Schwarz TL. 2002. Members of the synaptobrevin/vesicle associated membrane protein (VAMP) family in drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability. Proc Natl Acad Sci 99:13867-13872.
Lin W, Burgess RW, Dominguez B, Pfaff S, Sanes JR, Lee K-F. 2001. Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 410:1057-1064.
Terrado J, Burgess RW, DeChiara T, Yancopoulos G, Sanes JR, Kato AC. 2001. Motoneuron survival is enhanced in the absence of neuromuscular junction formation in embryos. J Neurosci 21:3144-3150.
Burgess RW, Skarnes WC, Sanes JR. 2000. Agrin isoforms with distinct amino-termini: differential expression, localization, and function. J Cell Biol 151:41-52.
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