Endoplasmic Reticulum Membrane Contact Sites Research Articles

Keep in contact: multiple roles of endoplasmic reticulum-membrane contact sites and the organelle interaction network in plants.

Functional regulation and structural maintenance of the different organelles in plants contribute directly to plant development, reproduction and stress responses. To ensure these activities take place effectively, cells have evolved an interconnected network amongst various subcellular compartments, regulating rapid signal transduction and the exchange of biomaterial. Many proteins that regulate membrane connections have recently been identified in plants, and this is the first step in elucidating both the mechanism and function of these connections. Amongst all organelles, the endoplasmic reticulum is the key structure, which likely links most of the different subcellular compartments through membrane contact sites (MCS) and the ER-PM contact sites (EPCS) have been the most intensely studied in plants. However, the molecular composition and function of plant MCS are being found to be different from other eukaryotic systems. In this article, we will summarise the most recent advances in this field and discuss the mechanism and biological relevance of these essential links in plants.

Altered Lysosomal Function Manipulates Cellular Biosynthetic Capacity By Remodeling Intracellular Cholesterol Distribution.

Lysosomes are known to influence cholesterol trafficking into endoplasmic reticulum (ER) membranes. Though intracellular cholesterol levels are known to influence the lipid biosynthetic responses in ER, the specific effects of lysosomal modulation on these outcomes is not known. To demonstrate this, C2C12 cells were treated with chloroquine, a lysosomotropic agent, and its effects on cellular biosynthetic capacity, structural and functional status of ER was determined. In addition to its known effects on autophagy reduction, chloroquine treatment induced accumulation of total cellular lipid and ER-specific cholesterol content. It was also observed that chloroquine caused an increase in smooth-ER content with defects in overall protein turnover. Further, since ER and mitochondria function in close association through ER membrane contact sites, it is likely that lysosomal modulation also brings about associated changes in mitochondria. In this regard, we found that chloroquine reduces mitochondrial membrane potential and mitochondrial dynamics. Collectively, the differential biosynthetic response of rise in lipid content, but not protein content, cannot be accounted by merely considering that chloroquine induced suppression of autophagy causes defects in organelle function. In this defective autophagy scenario, both biosynthetic responses such as lipid and protein synthesis are expected to be reduced rather than only the latter, as observed with chloroquine. These findings suggest that cholesterol trafficking/distribution within cellular organelles could act as an intracellular mediator of differential biosynthetic remodelling in interconnected organelles.

ER membrane contact sites support endosomal small GTPase conversion for exosome secretion.

Exosomes are endosome-derived extracellular vesicles involved in intercellular communication. They are generated as intraluminal vesicles within endosomal compartments that fuse with the plasma membrane (PM). The molecular events that generate secretory endosomes and lead to the release of exosomes are not well understood. We identified a subclass of non-proteolytic endosomes at prelysosomal stage as the compartment of origin of CD63 positive exosomes. These compartments undergo a Rab7a/Arl8b/Rab27a GTPase cascade to fuse with the PM. Dynamic endoplasmic reticulum (ER)-late endosome (LE) membrane contact sites (MCS) through ORP1L have the distinct capacity to modulate this process by affecting LE motility, maturation state, and small GTPase association. Thus, exosome secretion is a multi-step process regulated by GTPase switching and MCS, highlighting the ER as a new player in exosome-mediated intercellular communication.

VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites.

RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and invivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.

ER membrane contact sites: key platforms for biogenesis of RNA-containing extracellular vesicles.

The mechanisms by which cytoplasmic cargoes such as RNAs are incorporated into extracellular vesicles (EVs) are poorly understood. In a recent article published in Developmental Cell, we describe a novel function of endoplasmic reticulum membrane contact sites (ER MCS) in regulating biogenesis of RNA-containing EVs (Barman et al., 2022). We identified the ER MCS tether protein VAP-A and the ceramide transporter CERT as key drivers of this process. VAP-A depletion and overexpression produced corresponding changes in the overall number and RNA content of secreted EVs. Further sub-fractionation of small EVs from VAP-A depleted cells revealed a distinct loss in a specific subset of dense, RNA-loaded small EVs that are critical for the transfer of miR-100 to recipient cells. Cell imaging data confirmed the loss of RNA and RNA binding proteins (RBPs) in VAP-A-knockdown multivesicular bodies. Lipid analysis of VAP-A-knockdown EVs revealed decreases in ceramides, which are known to affect EV biogenesis. Depletion of the ceramide transfer protein CERT, which interacts with its binding partner VAP-A at ER MCS, leads to similar defects in EV number and RNA content as VAP-A-knockdown. These data suggest a model for ER MCS as platforms for biogenesis of a key EV population via ceramide transfer and RNA loading.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

SummaryThere is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

The journey of Ca2+ through the cell - pulsing through the network of ER membrane contact sites.

Calcium is the third most abundant metal on earth, and the fundaments of its homeostasis date back to pre-eukaryotic life forms. In higher organisms, Ca2+ serves as a cofactor for a wide array of (enzymatic) interactions in diverse cellular contexts and constitutes the most important signaling entity in excitable cells. To enable responsive behavior, cytosolic Ca2+ concentrations are kept low through sequestration into organellar stores, particularly the endoplasmic reticulum (ER), but also mitochondria and lysosomes. Specific triggers are then used to instigate a local release of Ca2+ on demand. Here, communication between organelles comes into play, which is accomplished through intimate yet dynamic contacts, termed membrane contact sites (MCSs). The field of MCS biology in relation to cellular Ca2+ homeostasis has exploded in recent years. Taking advantage of this new wealth of knowledge, in this Review, we invite the reader on a journey of Ca2+ flux through the ER and its associated MCSs. New mechanistic insights and technological advances inform the narrative on Ca2+ acquisition and mobilization at these sites of communication between organelles, and guide the discussion of their consequences for cellular physiology.

Interplay between endoplasmic reticulum membrane contacts and actomyosin cytoskeleton.

Eukaryotic membrane-bound organelles, exhibiting distinctive morphologies, dynamics and functions, are interconnected at membrane contact sites (MCSs) through numerous tethering machineries. MCSs are required for many fundamental cellular processes, such as non-vesicular lipid transfer, calcium transport and organelle homeostasis. Actin cytoskeleton and myosin motors are known to dynamically interact with different membrane boundaries, facilitating organelle movements and partitioning. Intriguingly, recent studies have pinpointed a special participation of actomyosin at various MCSs involving the endoplasmic reticulum (ER), the most extensive membranous organelle in the cell. Here, I summarize emerging roles of ER MCSs in modulating actomyosin structures and discuss feedback functions of such actomyosin regulation at these MCSs.

Light microscopy of the endoplasmic reticulum-membrane contact sites in plants.

The existence of membrane contact sites (MCS) has been reported in different systems in the past decade, and their importance has been recognised by the cell biology community. Amongst all endomembrane structures, the endoplasmic reticulum (ER) plays vital roles in organising the organelle interaction network with the plasma membrane (PM), Golgi bodies, mitochondria, plastids, endosomes and autophagosomes. A number of methods have been used to study the establishment and functions of these interactions, among them, light microscopy appears to be one of the most effective approaches. Here, we present an overview of the discovery of ER-PM contact sites, and highlight the latest developments in light microscopical-based techniques that can be used for their study.

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP‐A and VAP‐B, interact with proteins from other organelles that possess a small VAP‐interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain‐containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro. MOSPD2 is an ER‐anchored protein, and it interacts with several FFAT‐containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle‐bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.

GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells.

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

Structure and function of ER membrane contact sites with other organelles.

The endoplasmic reticulum (ER) is the largest organelle in the cell, and its functions have been studied for decades. The past several years have provided novel insights into the existence of distinct domains between the ER and other organelles, known as membrane contact sites (MCSs). At these contact sites, organelle membranes are closely apposed and tethered, but do not fuse. Here, various protein complexes can work in concert to perform specialized functions such as binding, sensing and transferring molecules, as well as engaging in organelle biogenesis and dynamics. This Review describes the structure and functions of MCSs, primarily focusing on contacts of the ER with mitochondria and endosomes.

Cell Cycle-dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells

The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at one or more sites located on its large cytoplasmic C terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.

Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals

Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca(2+) but their relationship to the better-characterised endoplasmic reticulum (ER) Ca(2+) store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca(2+) signals in primary cultured human fibroblasts. These Ca(2+) signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca(2+) oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca(2+) stores is thus sufficient to evoke ER-dependent Ca(2+) release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca(2+) mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca(2+) stores important for the genesis of Ca(2+) signal complexity.

Nucleus–vacuole junctions in yeast: anatomy of a membrane contact site

NV junctions (nucleus-vacuole junctions) in Saccharomyces cerevisiae are MCSs (membrane contact sites) formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the outer nuclear membrane, which is continuous with the perinuclear ER (endoplasmic reticulum). NV junctions mediate a unique autophagic process that degrades portions of the yeast nucleus through a process called 'piecemeal microautophagy of the nucleus' (PMN). Our studies suggest that the lipid composition of NV junctions plays an important role in the biogenesis of PMN structures. NV junctions represent a unique model system for studying the biology of ER MCSs, as well as the molecular mechanism of selective microautophagy.