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Imaging Endogenous PSD-95 MAGUKs in Hippocampal Neurons


Ochan Kwon Photo

Wake Forest University (North Carolina)

Abstract

Dendritic synapses within the hippocampus are critical for the conduction of electrochemical signals between neuronal synaptic junctions, thus allowing constant communication between neurons through neurotransmitter release. This signaling process requires the establishment of excitatory and inhibitory synapses. The more abundant excitatory glutamatergic synapses are characterized by a specialized macromolecular structure at the postsynaptic membrane called the postsynaptic density (PSD) located at the tip of dendritic spines. The PSD harbors functional molecules and receptors important for signaling transmission, processing and memory formation. A prominent protein at the PSD is PSD-95, a major scaffolding protein of the membrane-associated guanylate kinase (MAGUK) family localized in the excitatory postsynaptic sites. PSD-95 proteins are thought to be involved with synaptic plasticity and the maturation of dendritic spines. Furthermore, PSD-95 contains PDZ domains (protein-interaction domains) that allow it to form large molecular complexes with key PSD proteins such as adhesion molecules and other scaffolding proteins like GKAP and Shank. Lastly, PDZ domains in PSD-95 organize glutamate receptors such as NMDA and AMPA receptors and their associated signaling proteins, giving PSD-95 a critical role as a regulator of synaptic strength. Here we co-transfect PSD-95 and myosin-V intrabody constructs in rat hippocampal neurons, which allows live labeling of endogenous PSD-95 and myosin V and direct imaging of both endogenous proteins located within synapses and neuronal processes. In DVI 5-14 hippocampal neurons, after ~ 20 hr expression, time lapse imaging shows that most of the PSD-95 puncta are motionless along dendrite processes while some at the cell body and distal dendrites show significant movement.  Dual channel imaging of PSD-95 and myosin V shows co-movement of both puncta at ~ 0.1-0.3 um/s, matching the known speed of myosin V motor in vitro. Immuno-gold labeling of GFP tag from the PSD-95 intrabody for electron microscopy showed that labels localized at the PSDs and at vesicles in neuronal processes. This imaging data demonstrates that myosin V is likely the molecular motor involved in transporting PSD-95 in neurons.

Last updated November 27, 2013