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Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Famatta Perry - Delaware State University
Co-Author(s): Murali Temburni, Delaware State University, Dover, DE
The specification of neurites into axons is essential for the differentiation of a vertebrate neuron. However, the mechanisms as well as the main proteins responsible have not yet been identified. Adenomatous polyposis coli (APC), a multifunctional protein has been shown to play a role by transporting the mPar3/6 complex to the neurite that ultimately becomes the axon. APC is localized to the post-synaptic density and interacts with scaffolding and signaling proteins to organize nicotinic cholinergic synapses in the vertebrate Ciliary Ganglion (CG). Results from our previous research demonstrate that post synaptic density protein, PSD93 interacts with APC. However, this interaction is not necessary for synapse formation. APC and PSD93 are also co-localized extra-synaptically on the neuronal membrane, particularly on the axon initial segment. We hypothesize that the function of the APC-PSD93 complex is to determine neuronal polarity and the subsequent specialization of a single neurite into the axon. This hypothesis is based on the evidence that APC is needed for localization of the mPar6 neuronal polarity complex to the incipient neurite and PSD93 is localized to the axon initial segment. We are testing this hypothesis using a dominant-negative (DN) strategy, to block APC-PSD93 interactions specifically without affecting other functions. In the first phase of this research, our aim is to analyze APC’s interaction with PSD93 in chick embryo ciliary ganglia neurons cultured in vitro. We have generated the APC:PSD93 DN construct which expresses the C-terminal PDZ binding domain of APC in the K6205 lentiviral vector. In this study, E7 chick embryo CG neurons are infected with APC:PSD93 DN virus or control empty K2605 virus in culture. Neurite extension is monitored by phase contrast microscopy with Nomarski optics and infected neurons are tracked by GFP expression. Our initial results indicate that when APC: PSD93 interactions are disrupted, axon determination, but not neurite outgrowth, is disrupted. Next, we are assaying the localization of endogenous APC, PSD93 and the dominant negative peptides by immunofluorescence using specific antibodies. In the second phase of our research, we will evaluate the influence of APCPSD93 interactions in embryonic chick brain in vivo. We will introduce the APC:PSD93 dominant negative expressing and control lentiviruses into 36 hour chick embryos through microinjections. Ciliary ganglion and optic tectum neurons of E7 to E12 embryos will be harvested from the injected embryos and prepared for immunofluorescence with specific antibodies. We expect that the APC:PSD93 dominant negative construct will prevent axon specification in the chick ciliary ganglion as well as optic tectum neurons.
Funder Acknowledgement(s): NSF HBCU-UP HRD-1533631
Faculty Advisor: Murali Temburni,
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