THESIS
2010
xi, 60 p. : ill. (shiefly col.) ; 30 cm
Abstract
The neuromuscular junction (NMJ) is the synapse located at the interface between a motor axon terminal and a muscle fiber. During NMJ formation, acetylcholine receptors (AChRs) in muscle become clustered to an extremely high density in the postsynaptic membrane. This aggregation of AChRs, which is a hallmark of postsynaptic differentiation, depends on the activation of the muscle-specific tyrosine kinase MuSK through its interaction with agrin secreted by the motor axon and is regulated by other muscle-intrinsic tyrosine kinases and phosphatases. Src is a ubiquitous tyrosine kinase that has been shown to play a role in the formation of AChR clusters....[
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The neuromuscular junction (NMJ) is the synapse located at the interface between a motor axon terminal and a muscle fiber. During NMJ formation, acetylcholine receptors (AChRs) in muscle become clustered to an extremely high density in the postsynaptic membrane. This aggregation of AChRs, which is a hallmark of postsynaptic differentiation, depends on the activation of the muscle-specific tyrosine kinase MuSK through its interaction with agrin secreted by the motor axon and is regulated by other muscle-intrinsic tyrosine kinases and phosphatases. Src is a ubiquitous tyrosine kinase that has been shown to play a role in the formation of AChR clusters.
Works previously published by our lab showed that activated src was localized at AChR clusters. To further explore the src activation in live cells, we utilized the membrane-targeted src reporter based on the fluorescence resonance energy transfer (FRET) technique to visualize this process in live cells. The reporter consists of an SH2 domain and a specific substrate peptide with CFP and YFP flanking the N and C termini of the complex. Phosphorylation of the substrate peptide by src changes the reporter’s FRET signal.
The specificity of membrane-targeted src reporter was tested in HEK 293T cells. The starved cells showed a remarkable increase of CFP/YFP ratio with bFGF treatment, which implicated the increase of src activation. And this increase can be inhibited by selective src family inhibitor PP1 analogue or FGF receptor inhibitor SU5402. Thus, bFGF stimulation led to src activation through FGF receptor-mediated pathway.
By expressing the membrane-targeted reporters in live Xenopus muscle cells, the temporal src activation in NMJ induction was examined. A rapid increase of src activation was detected in muscle cells after agrin or bFGF treatment. To magnify the activation, tyrosine phosphatase inhibitor pervanadate (PV) was used. This allowed the visualization of a faster and stronger of src activation in muscle cells in response to agrin treatment. Besides, all the increase of src activation can be inhibited by PP1. These data revealed the temporal src activation in the early stage of NMJ induction.
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