The Role of ESyt2 Phosphorylation in the Regulation of ER-Plasma Membrane Contact Sites in Mitosis.

Abstract

Membrane contact sites (MCS) are regions of close apposition between intracellular organelles (< 30nm), maintained by tether proteins that physically bridge and stabilize the junctions. These sites regulate signaling and inter-organelle communication, and defects are implicated in pathologies such as neurodegeneration, cancer, and aging. Endoplasmic reticulum (ER)-plasma membrane contact sites (ERPMCS) were among the first MCS discovered and are involved in crucial cellular processes, including lipid exchange, calcium signaling, and membrane remodeling. Recent studies indicate that ERPMCS are dramatically downregulated during mitosis. This downregulation regulates Ca2+ influx into the cell, which is inhibited during mitosis due to ERPMCS loss. However, the mechanisms involved remain unclear. I hypothesize that that phosphorylation of tether proteins that maintain the integrity of ERPMCS is involved in their downregulation in mitosis. Several ERPMCS tethers have been shown to be phosphorylated in mitosis, including Extended Synaptotagmin 2 (ESyt2). In this study I directly test whether ESyt2 phosphorylation in mitosis is involved in the downregulation of ERPMCS. I generated alanine substitution mutants of ESyt2 where all the Ser/Thr residues shown to be phosphorylated in mitosis are mutated to Ala. I also generate a phosphomimetic mutant where the residues are mutated to Glu. Expression of the ESyt2-E mutant shows a trend toward reducing ERPMCS in interphase in cells where the endogenous ESyt2 gene has been knocked out. Importantly, expression of ESyt2-A during mitosis partially reverses the loss of ERPMCS. These data support my hypothesis and provide critical insights into the mechanism controlling ERPMCS during cell division.

Date of Award	2025
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences