First Thesis Advisor
Justin R. Fallon
Neuromuscular Junction, Agrin, Synaptic Transmission
The nervous system requires rapid, efficient, and accurate transmission between cells for proper functioning. Synapses are the predominant structures through which such vital communication occurs. How synapses are formed, maintained, and eliminated are questions of fundamental importance.
At the nerve-muscle synapse, formation of the postsynaptic apparatus is directed by agrin. The hallmark activity of agrin is the aggregation of acetylcholine receptors (AChRs) into dense clusters opposite the presynaptic nerve terminal. Early events in the agrin signal transduction cascade include activation of the receptor tyrosine kinase MuSK and tyrosine phosphorylation of AChRs, but how these events lead to AChR cluster formation is unknown. Using the calcium buffer BAPTA, we demonstrate that intracellular calcium fluxes are necessary for agrin-induced formation of AChR clusters. However, clamping calcium fluxes before agrin stimulation does not alter agrin-induced phosphorylation of either MuSK or AChRs, indicating that this calcium-dependent step occurs downstream of both MuSK and AChR phosphorylation. These results identify a new step in the agrin signaling pathway required for the formation of AChR clusters.
We show that intracellular calcium fluxes also play an important role in stabilizing AChR clusters. Clamping intracellular calcium fluxes results in rapid dispersal of AChR clusters and dephosphorylation of both MuSK and AChRs, even if agrin is continually present. Furthermore, the protein tyrosine phosphatase inhibitor pervanadate inhibits both the dispersal and dephosphorylation, indicating a role for a tyrosine phosphatase in AChR cluster dispersal. Our data indicate that AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes that tonically inhibit a tyrosine phosphatase localized to AChR clusters. Our findings also show that distinct molecular mechanisms mediate the formation and the dispersal of agrin-induced AChR clusters.
The work presented here expands our understanding of synaptic differentiation in several ways. First, I characterized a new, calcium-dependent step required for the formation of agrin-induced AChR clusters. Next, I showed that postsynaptic specializations must be actively maintained, and describe a molecular mechanism that stabilizes AChR clusters. Finally, dispersal and formation of AChR clusters occurs by distinct pathways. Our understanding of the mechanisms regulating the formation and modulation of synapses will help us to better understand how the nervous system develops and responds to the world around us.
Megeath, LJ. Characterization of the Molecular Mechanisms Regulating the Agrin Signaling Pathway: a Dissertation. (1999). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 29. DOI: 10.13028/8anh-ja80. https://escholarship.umassmed.edu/gsbs_diss/29
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