Abstract
Super-resolution light and electron microscopy have revolutionized our ability to visualize cell machinery. We use live-cell super-resolution imaging including stimulated emission depletion (STED) microscopy together with highly inclined thin illumination (HiLo) and high-speed three-dimensional widefield imaging to visualize organelle dynamics in real time. We have integrated these approaches with in situ cryo-electron tomography (cryo-ET), and cryo-correlative light and electron microscopy (cryo-CLEM) to visualize the endoplasmic reticulum (ER) and its relationships with other intracellular organelles in near-native states. The combination of these methods has revealed a novel ER-derived compartment that is mobile, vesicular, and associated with mammalian 80S cytoplasmic ribosomes. Moreover, with cryo-focused-ion-beam (FIB) milling and cryo-ET, we show that these vesicles exist as discrete structures separate from the intact reticular ER architecture. We call these organelles Ribosome-Associated Vesicles (RAVs). Detailed characterization of the RAVs revealed that these structures are conserved across multiple cell types and species using both conventional transmission electron microscopy (TEM) and cryo-ET.
*Louis V. Gerstner Jr., Scholars Program (Z.F.), the Leon Levy Foundation (Z.F.), the John F. and Nancy A. Emmerling Fund of The Pittsburgh Foundation (Z.F.), Department of Defense PR141292 (Z.F.), NIH K08DA031241 (Z.F.), NSFMCB-1408986 (S.A.M.), National Science Foundation Graduate Research Fellowship (N.H.T.), NIH K01AG045335-01A1 (E.A.G.), NIH 1S10RR019003 (S.C.W.), NIH 1S10RR025488 (S.C.W.), NIH 1S10RR016236 (S.C.W.), the Howard Hughes Medical Institute (P.W., N.H.T., J.F., G.J.J.), NIH GM29169 (J.F.), Israel Science Foundation Grant 1285/14 (S.G.W.), European Research Council under the European Union’s Seventh Framework Programme [310649] (D.F.), NIH P50 GM082545 (G.J.J.), MINECO AIC-A-2011-0638 (J.M.C.), BIO2016-76400-R AEI/FEDER, UE (J.M.C.).
