The delicate balance between endocytosis and recycling of the cell surface receptors (NMDAR and AMPAR) is essential for controlling their surface levels and degradation, and is regulated by numerous processes including lateral membrane diffusion, scaffolding protein interactions and posttranslational modifications. Generally the NMDARs undergo activity-dependent endocytosis within clathrin-coated vesicles. They then enter the endosomal system where they are either sorted into the degradative lysosomal pathway, or are replenished via endosomal recycling. Defects in endosomal trafficking therefore lead to perturbed homeostasis of NMDARs. Our recent findings provide a comprehensive understanding of how post-translational modifications of NMDAR define an extended electrostatic peptide code for cargo sorting and influence their interactions with the trafficking machinery. Currently, I am trying to understand the mechanistic basis of intracellular trafficking in NMDAR receptor homeostasis. In my talk, I will be discussing about some of our efforts in the basic studies of the structure and function of SNX27, a unique member of PX-FERM module, that control membrane trafficking. Additionally, I will highlight the novel role for phosphorylation of the NMDARs in promoting SNX27-retromer interactions, which may have significant implications for activity-dependent trafficking of NMDARs during synaptic potentiation.

Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we employed high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.

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Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsPbinding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi-and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P 3 -enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.