This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We recently found that a synaptic membrane protein Synaptotagmin (Syt) binds a kinesin motor protein adaptor called D76 in a phosphorylation-dependent manner in a biochemical experiment (co-precipitation assay). We would like to validate this data via FRET, by using CFP-Syt and YFP-D76, in the presence and absence of the kinase activity. We assume that the kinase activity would bring the two proteins together, hence getting FRET signal, but what we don't know is how long this interaction would last or whether a de-phosphorylation would trigger disassembly of the protein complexes. Thus, we would like to analyze the dynamics of membrane cargo - transport motor affinity, in particular, whether the phospho-reaction would indeed be a triggering signal for cargo-motor assembly or disassembly. We believe the live imaging using FRET is the best way of addressing this question. Project restarted for a new set of constructs.
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