title: Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum-plasma membrane contact sites.
abstract: The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca(2+) homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt-mediated ER-PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt-dependent contacts were by far the predominant contacts, ER-PM distance (19-22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca(2+) shortened the ER-PM distance at E-Syt1-dependent contacts sites. E-Syt-mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca(2+) channel Orai1 as well as store operated Ca(2+) entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt-dependent ER-PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.
authors: Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510.
authors: Rubén
authors: R
authors: Fernández-Busnadiego
authors: Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510.
authors: Yasunori
authors: Y
authors: Saheki
authors: Department of Cell Biology, Howard Hughes Medical Institute and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510 pietro.decamilli@yale.edu.
authors: Pietro
authors: P
authors: De Camilli
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D008565:Membrane Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D050857:Synaptotagmins / Q000378:metabolism*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D050857:Synaptotagmins; D002118:Calcium
keywords: E-Syt; TULIP; cryo-electron microscopy; lipid transfer; phosphoinositides