bims-mecosi 2022-03-06 papers

Issue of 2022–03–06
six papers selected by
Verena Kohler, Stockholm University

J Biol Chem. 2022 Feb 26. pii: S0021-9258(22)00220-4. [Epub ahead of print] 101780

In Situ Artificial Contact Sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein tethering activities.

Julie Milanini, Maud Magdeleine, Nicolas Fuggetta, Souade Ikhlef, Frédéric Brau, Sophie Abelanet, Fabien Alpy, Catherine Tomasetto, Guillaume Drin.

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum (ER) via the specific interaction of the FFAT motif with ER-resident vesicle-associated membrane protein-associated proteins (VAPs). This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins, and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3 (STARD3). Collectively, these data indicate that the creation of ISACS represents an efficient approach for studying the membrane tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

Keywords: Cell biology; Confocal microscopy; Endoplasmic reticulum (ER); FFAT motif; Lipid transport; Liposome; Membrane contact sites; OSBP; STARD3; VAP

DOI: https://doi.org/10.1016/j.jbc.2022.101780

Semin Cell Dev Biol. 2022 Feb 25. pii: S1084-9521(22)00058-1. [Epub ahead of print]

Molecular machinery regulating organelle dynamics during axon growth and guidance.

Michiko Shirane, Hiroyuki Kamiguchi.

Axon growth and guidance in the developing nervous system rely on intracellular membrane dynamics that involve endosome maturation and transport, as well as its regulated tethering to the endoplasmic reticulum (ER). Recent studies have identified several key molecules, such as protrudin, which plays a dynamic role at membrane contact sites between the ER and endosomes/lysosomes, and myosin Va, which acts as a sensor for ER-derived Ca2+ that triggers peri-ER membrane export. These molecules form different types of multiprotein complexes at the interface of organelles and, in response to their surrounding microenvironments, such as Ca2+ concentrations and lipid contents, regulate the directional movement of endosomal vesicles in extending axons. Here, we review the molecular mechanisms underlying membrane dynamics and inter-organelle interactions during neuronal morphogenesis.

Keywords: Calcium; Endoplasmic reticulum; Endosome; Membrane contact site; Myosin Va; Protrudin

DOI: https://doi.org/10.1016/j.semcdb.2022.02.019

PLoS Genet. 2022 Mar 03. 18(3): e1010106

ER-PM membrane contact site regulation by yeast ORPs and membrane stress pathways.

Evan Quon, Aleksa Nenadic, Mohammad F Zaman, Jesper Johansen, Christopher T Beh.

In yeast, at least seven proteins (Ice2p, Ist2p, Scs2/22p, Tcb1-Tcb3p) affect cortical endoplasmic reticulum (ER) tethering and contact with the plasma membrane (PM). In Δ-super-tether (Δ-s-tether) cells that lack these tethers, cortical ER-PM association is all but gone. Yeast OSBP homologue (Osh) proteins are also implicated in membrane contact site (MCS) assembly, perhaps as subunits for multicomponent tethers, though their function at MCSs involves intermembrane lipid transfer. Paradoxically, when analyzed by fluorescence and electron microscopy, the elimination of the OSH gene family does not reduce cortical ER-PM association but dramatically increases it. In response to the inactivation of all Osh proteins, the yeast E-Syt (extended-synaptotagmin) homologue Tcb3p is post-transcriptionally upregulated thereby generating additional Tcb3p-dependent ER-PM MCSs for recruiting more cortical ER to the PM. Although the elimination of OSH genes and the deletion of ER-PM tether genes have divergent effects on cortical ER-PM association, both elicit the Environmental Stress Response (ESR). Through comparisons of transcriptomic profiles of cells lacking OSH genes or ER-PM tethers, changes in ESR expression are partially manifested through the induction of the HOG (high-osmolarity glycerol) PM stress pathway or the ER-specific UPR (unfolded protein response) pathway, respectively. Defects in either UPR or HOG pathways also increase ER-PM MCSs, and expression of extra "artificial ER-PM membrane staples" rescues growth of UPR mutants challenged with lethal ER stress. Transcriptome analysis of OSH and Δ-s-tether mutants also revealed dysregulation of inositol-dependent phospholipid gene expression, and the combined lethality of osh4Δ and Δ-s-tether mutations is suppressed by overexpression of the phosphatidic acid biosynthetic gene, DGK1. These findings establish that the Tcb3p tether is induced by ER and PM stresses and ER-PM MCSs augment responses to membrane stresses, which are integrated through the broader ESR pathway.

DOI: https://doi.org/10.1371/journal.pgen.1010106

Front Physiol. 2021 ;12 791691

Transmission Electron Microscopy to Follow Ultrastructural Modifications of Erythroblasts Upon ex vivo Human Erythropoiesis.

Alice Dussouchaud, Julieta Jacob, Charles Secq, Jean-Marc Verbavatz, Martina Moras, Jérôme Larghero, Claudio M Fader, Mariano A Ostuni, Sophie D Lefevre.

Throughout mammal erythroid differentiation, erythroblasts undergo enucleation and organelle clearance becoming mature red blood cell. Organelles are cleared by autophagic pathways non-specifically targeting organelles and cytosolic content or by specific mitophagy targeting mitochondria. Mitochondrial functions are essential to coordinate metabolism reprogramming, cell death, and differentiation balance, and also synthesis of heme, the prosthetic group needed in hemoglobin assembly. In mammals, mitochondria subcellular localization and mitochondria interaction with other structures as endoplasmic reticulum and nucleus might be of importance for the removal of the nucleus, that is, the enucleation. Here, we aim to characterize by electron microscopy the changes in ultrastructure of cells over successive stages of human erythroblast differentiation. We focus on mitochondria to gain insights into intracellular localization, ultrastructure, and contact with other organelles. We found that mitochondria are progressively cleared with a significant switch between PolyE and OrthoE stages, acquiring a rounded shape and losing contact sites with both ER (MAM) and nucleus (NAM). We studied intracellular vesicle trafficking and found that endosomes and MVBs, known to be involved in iron traffic and heme synthesis, are increased during BasoE to PolyE transition; autophagic structures such as autophagosomes increase from ProE to OrthoE stages. Finally, consistent with metabolic switch, glycogen accumulation was observed in OrthoE stage.

Keywords: autophagy; electron microscopy; erythropoiesis; mitochondria; vesicles

DOI: https://doi.org/10.3389/fphys.2021.791691

Virology. 2022 Feb 26. pii: S0042-6822(22)00035-6. [Epub ahead of print]569 29-36

Lipid metabolism is involved in the association of rotavirus viroplasms with endoplasmic reticulum membranes.

José L Martínez, Catherine Eichwald, Elisabeth M Schraner, Susana López, Carlos F Arias.

Rotavirus (RV) replication occurs in cytoplasmic membrane-less, electron-dense inclusions termed viroplasms, composed of viral and cellular elements. These inclusions have been shown to colocalize with components of the lipid droplets (LDs), unique organelles that play an essential role in lipid metabolism. Given the robust LDs-viroplasm association, LDs have been proposed to serve as a scaffold for viroplasm assembly. Interestingly, no evidence has described the participation of lipid metabolism in other RV replication steps. Here, we report that lipid metabolism is essential to maintain the production of the infectious virus through a process independent of viroplasm biogenesis. Disruption of the lipogenesis-lipolysis balance dissociates endoplasmic reticulum membranes from viroplasms, suggesting that lipid metabolism is essential for a continuous flux of lipids to allow the association between viroplasms and ER membranes. LDs could also be relevant as lipid reservoirs for membrane synthesis required to form mature infectious rotavirus particles.

Keywords: Lipid droplets; Lipogenesis; Lipolysis; Rotavirus; Viroplasms

DOI: https://doi.org/10.1016/j.virol.2022.02.005