Extended‐Synaptotagmins Modulate Diacylglycerol Dynamics at the Immunological Synapse in Human T Lymphocytes

Abstract

Cytotoxic T‐lymphocytes (CTLs) regulate the immune response by recognizing and destroying cancerous or virus‐infected cells through the polarized release of apoptosis‐inducing proteins at the contact area known as the immunological synapse (IS). The synaptic membrane is a heterogenous region where protein complexes rearrange on the cell surface. Within the membrane are phospholipids that assemble to relay information from extracellular receptors to induce global cellular changes. Diacylglycerol (DAG) is a key secondary messenger of T‐cell receptor (TCR) activation which induces microtubule‐organizing center polarization towards the IS and signaling via MAPK and NFkB pathways. Abnormal lipid homeostasis at the synapse is linked to autoimmune disorders. However, our understanding on how cells regulate dynamic transfer of lipids from one cellular compartment to another is still scant. Evidence points to the role of lipid transport proteins (LTPs) in the regulation of lipid homeostasis without the need for membrane fusion. Extended‐Synaptotagmins (E‐Syts) are a group of LTPs tethering the endoplasmic reticulum (ER) to plasma membrane (PM), thus controlling membrane lipid compositions by transporting DAG down its concentration gradient. Herein, we tested the hypothesis that E‐Syts play a role in the modulation of diacylglycerol at the synaptic membrane, ER‐PM morphology and signaling dynamics by performing the following experiments: 1) assessing the role of E‐Syts in regulating the effector functions of human lymphocytes by measuring cellular cytotoxicity, degranulation, TCR signaling via phospho‐protein analysis and intracellular Ca2+ levels; 2) testing the role of E‐Syts in DAG distribution at the IS via stimulated emission depletion (STED) microscopy. Our results indicate that E‐Syts down‐modulate human lymphocyte activation as their knock‐down leads to enhanced TCR signaling, degranulation, and cytokine production measured by flow cytometry. Using GFP‐based bioprobes for DAG and tagged E‐Syt constructs, we observed proximal localization of E‐Syts with signaling protein LAT and CD4 via super‐resolution microscopy. Accordingly, E‐Syt1 knockout Jurkat cells at resting state express localized DAG accumulation similar to activated WT Jurkat cells. Our results provide, for the first time, an understanding of spatio‐temporal distribution as well as the role of lipid regulators at membrane junction sites during IS formation. We believe that E‐Syts play an essential role in maintenance of synaptic lipids via regulation of non‐vesicular lipid transport, whose loss might contribute to enhanced activity of CTLs at resting conditions. Collectively, our results suggest that E‐Syts are critical components to orchestrate lipid distribution during synapse formation. Future experiments will be required to shed light on additional steady state regulators of synaptic membrane lipids, highlighting the importance of membrane crosstalk in the setting of CTLs.