Exocyst complex subunit sec8 binds to postsynaptic density protein-95 (PSD-95): a novel interaction regulated by cypin (cytosolic PSD-95 interactor).

The PDZ domains of postsynaptic density (PSD) protein-95 play a role in the localization of PSD-95 and binding partners to neuronal synapses. The identification of binding partners to these PDZ domains can help us in understanding how signalling complexes are assembled. We observed that one of the subunits in the sec6/8 or exocyst complex, sec8, contains a C-terminal consensus sequence for PDZ binding. Sec8 binds to PDZ1-2 of PSD-95, and this binding can be competed with a peptide that binds to PDZ1 and PDZ2 in the peptide-binding site. In addition, binding of sec8 is dependent on its C-terminal-binding sequence namely Thr-Thr-Val (TTV). Immunoblotting of rat tissue extracts shows that sec8 and PSD-95 are enriched in the same brain regions, and sec8 and PSD-95 have the same subcellular distribution in pheochromocytoma cells, suggesting that these proteins may interact in vivo. Immunoprecipitation studies of sec8 and PSD-95 in brain provide further evidence of a sec8 and PSD-95 interaction. Furthermore, the cytosolic PSD-95 interactor competes with sec8 for interaction with PSD-95. Taken together, our results suggest that the cytosolic PSD-95 interactor may function to regulate the ability of sec8 to bind to PSD-95

A Novel Role for Snapin in Dendrite Patterning: Interaction with Cypin

Temporal and spatial assembly of signal transduction machinery determines dendrite branch patterning, a process crucial for proper synaptic transmission. Our laboratory previously cloned and characterized cypin, a protein that decreases PSD-95 family member localization and regulates dendrite number. Cypin contains zinc binding, collapsin response mediator protein (CRMP) homology, and PSD-95, Discs large, zona occludens-1 binding domains. Both the zinc binding and CRMP homology domains are needed for dendrite patterning. In addition, cypin binds tubulin via its CRMP homology domain to promote microtubule assembly. Using a yeast two-hybrid screen of a rat brain cDNA library with cypin lacking the carboxyl terminal eight amino acids as bait, we identified snapin as a cypin binding partner. Here, we show by affinity chromatography and coimmunoprecipitation that the carboxyl-terminal coiled-coil domain (H2) of snapin is required for cypin binding. In addition, snapin binds to cypin's CRMP homology domain, which is where tubulin binds. We also show that snapin competes with tubulin for binding to cypin, resulting in decreased microtubule assembly. Subsequently, overexpression of snapin in primary cultures of hippocampal neurons results in decreased primary dendrites present on these neurons and increased probability of branching. Together, our data suggest that snapin regulates dendrite number in developing neurons by modulating cypin-promoted microtubule assembly

Retinal Laser Burn Disrupts Immune Privilege in the Eye

Immune privilege allows for the immune protection of the eye in the absence of inflammation. Very few events are capable of overcoming the immune-privileged mechanisms in the eye. In this study, we report that retinal laser burn (RLB) abrogates immune privilege in both the burned and nonburned eye. As early as 6 hours after RLB, and as late as 56 days after RLB, antigen inoculation into the anterior chamber of the burned eye failed to induce peripheral tolerance. After RLB, aqueous humor samples harvested from nontreated eyes but not from either the burned or the contralateral eye, down-regulated the expression of CD40 and up-regulated interleukin-10 mRNA in peritoneal exudate cells, and converted peritoneal exudate cells into tolerogenic antigen-presenting cells (APCs). Unlike F4/80+ APCs from nontreated mice, F4/80+ APCs from RLB mice were unable to transfer tolerance after anterior chamber inoculation of antigen into naïve mice. The increased use of lasers in both the industrial and medical fields raises the risk of RLB-associated loss of immune regulation and an increased risk of immune inflammation in the eye