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Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool.

ABSTRACT: Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.

SUBMITTER: Grimmer M

PROVIDER: S-EPMC7033103 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool.

Grimmer Mona M Bacia Kirsten K

Scientific reports 20200220 1

Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiol ...[more]

PMID: 32080222

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Project description:Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus-host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

Project description:Aim and experimental set-up: Sequencing of small RNA from total RNA fractions. The aim of this experiment was to compare profiles of miRNA expression between the following genetic backgrounds: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter). Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested, and total RNA was prepared by Trizol extraction. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform. Sequencing of small RNA bound to AGO1 in membrane fractions The aim of this experiment was to characterize AGO1-bound small RNAs in membrane fractions, and to answer two specific questions: Can miRNAs be identified whose association with membrane-bound AGO1 is different between the following four genotypes: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter) Is the ratio between reads matching miRNA and miRNA* different between the above four genotypes? Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested. Microsomes were prepared from 2g of seedling tissue, solubilized by 1% deoxycholate and AGO1 was immunoprecipitated with specific antibodies (Agrisera). Immunopurified AGO1 was eluted from Protein A sepharose beads by competitive elution with antigenic AGO1 peptide. Small RNA was extracted by Trizol extraction from eluted AGO1. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform.

Dataset Information

Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool.

Publications

Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool.

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