SUMMARY Most available information on ER-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca2+ entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here we show that the three Extended-Synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca2+ in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca2+ regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca2+ entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast), mediate the formation of ER-PM contacts sites which are functionally distinct from those mediated by STIM1 and Orai1.

Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase OCRL ), which acts on PI(4,5)P 2 and PI(3,4,5)P 3 , was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase OCRL to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P 2 and PI (3,4,5)P 3 biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase OCRL recruitment and PI(4,5)P 2 dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P 2 biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P 3 synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.endocytosis | polarity | rapamycin | ion channel | ruffles P hosphoinositides (PIs) are key signaling components of cell membranes that result from the reversible phosphorylation of phosphatidylinositol at the 3, 4, and 5 positions of the inositol ring. These reactions give rise to seven PI species that regulate a variety of cell processes including membrane trafficking, cytoskeleton dynamics, cell migration, cytokinesis, and ion and metabolite fluxes across membranes. Their signaling is linked to their different subcellular localization, rapid turnover, and distinct repertoire of binding proteins (1-3). Given these properties, functional interrogation of PI signaling through experimental manipulation requires precise spatial and temporal control.To date, most studies investigating the role of these lipids have relied on pharmacological or genetic perturbations of the enzymes responsible for PI synthesis and degradation. Small molecules that affect these enzymes have been developed, but, as is the case with all drugs, their off-target effects cannot be excluded. Genetic manipulations (knockout, knockdown, and overexpression studies), as well as studies of patients harboring mutations in such enzymes, have greatly advanced our understanding of the funct...