Regulation of calcium fluxes at endoplasmic reticulum – plasma membrane contact sites

Abstract

Calcium ions play an important role as secondary messenger in cell biology therefore, regulation of its cellular concentration is key to maintain homeostasis and trigger signaling events. A multitude of pathways have been developed during evolution to tightly control the in and out of cellular calcium ions. Store-operated calcium entry (SOCE) is a major signaling pathway involved in proper development of muscle cells and immune system activation. During SOCE, an agonist stimulation generates inositol trisphosphate (IP3) from phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis. Following, IP3 induces depletion of endoplasmic reticulum (ER) calcium store through IP3 receptor (IP3R). Stromal interaction protein 1 (STIM1), an ER transmembrane protein, senses calcium depletion through its luminal domains which induce a conformational change exposing key cytosolic domains. STIM1 new intramolecular arrangement favors its oligomerization and in turn its translocation to cortical ER (cER), which is closely apposed to plasma membrane (PM). Within those ER-PM junctions, STIM1 binds and gate ORAI1 calcium channel on the PM and induce calcium entry. Refilling of the ER store and cytosolic calcium elevations are responsible for SOCE termination due to loss of STIM-ORAI complexes though calcium dependent inactivation processes. Physical interaction between STIM1 and ORAI1 occurs at ER-PM junctions where the gap distance is permissive and ranges from 10 nm to 25 nm. ER-PM junctions are part of the membrane contact sites (MCS) family which are membranes in close apposition between different organelles or subcellular structures, with an intermembrane distance usually below 30 nm. Interestingly, ER-PM contact sites play an important role in sustaining calcium signaling through replenishment of PIP2 in the PM. MCS are dynamic structures using tether proteins to adapt to cellular needs and are involved in the shaping of physiological responses. During SOCE, cER length increases in a STIM1 dependent manner and global coverage of ER-PM junctions increases by ~5 fold. Meanwhile, ER-PM gap distance is shortened from 21.8 nm to 14.8 nm by recruitment of ESyt1, an ER resident tether protein activated by calcium entry. MCS structure and function have been dependent one to another, however, a unifying model is missing. The first part of my thesis focuses on understanding of the relationship between SOCE and ER-PM contact site ultrastructure. To this end, we modified morphometric parameters of ER-PM contact sites by expressing known tether proteins (ESyt1 and ESyt2) or synthetic constructs from MAPPER family (MAPPER-Short and MAPPER-Long). Using electron microscopy (EM), we assessed ER-PM contact morphology upon expression of tether proteins. cER length and its associated ER-PM distance have been systematically measured and correlated at different time points of SOCE. Both parameters combined revealed that ER depletion is increasing cER length from 66 nm to 156 nm without significantly impacting gap distance. On the other hand, expression of tether proteins amplifies cER elongation upon calcium depletion. It is particularly dramatic with ESyt2 and MAPPER-Long which generate contacts longer than 1.5 µm. Additionally, these long contact sites were stabilized to an ER-PM distance specific to each tether family. As ESyt2 and MAPPER-L behave similarly at morphometric level, they were associated with a ~50% reduction of calcium entry in multiple cellular systems. These results support a role for ER-PM ultrastructure in regulation of SOCE through recruitment of tether proteins. Microscopy imaging is part of the core techniques used in the study of calcium signaling and MCS. Generation of high number of images represents new challenges in the way we deal with information and pipelines are now more and more essential for a smooth and reliable data processing. New approaches are developed every day to increase workflow from acquisition to extraction of key features from data. Due to the high amount of data generated in this thesis I included as a second part the development of new analytic tools to create a powerful and reliable pipeline for calcium, TIRF and ER-PM junction analysis. The objectivity and time saving opportunities offered by this pipeline are substantial and its reliability makes it used by most of my co-workers and even some collaborators. I additionally generated custom strategies to overcome limitation we were facing in data analysis of our microscopy timelapses. We could extract information that would have been out of reach without help from these analytic methods. Overall, using electron microscopy and calcium imaging with my pipeline I can conclude that cER expansion during SOCE is mediated by STIM1 and is enhanced by tether proteins recruited to contact sites. This might have potential application for regulation of SOCE during immune cell activation and open the way to a better understanding of how cells regulate their signaling through the MCS ultrastructure.