Network Organisation and the Dynamics of Tubules in the Endoplasmic Reticulum

ABSTRACTThe endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed a classification system for ER dynamics in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active reorganisation and dynamics were observed. Live cell ER tubule dynamics were classified using a Gaussian mixture model, defining tubule motion as active or thermal and conformational phase space analysis allowed this classification to be refined by tubule curvature states.STATEMENT OF SIGNIFICANCEThe endoplasmic reticulum (ER), a subcellular organelle, is an underexplored real-world example of active matter. Many processes essential to cell survival are performed by the ER, the efficacy of which may depend on its organisation and dynamics. Abnormal ER morphology is linked to diseases such as hereditary spastic paraplegias and it is possible that the dynamics are also implicated. Therefore, analysing the ER network in normal cells is important for the understanding of disease-related alterations. In this work, we outline the first thorough quantification methods for determining ER organisation and dynamics, deducing that tubule motion has a binary classification as active or thermal. Active reorganisation and dynamics along with indications of tension imbalances and membrane contact sites were observed.

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Network organisation and the dynamics of tubules in the endoplasmic reticulum

Scientific Reports ◽

10.1038/s41598-021-94901-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hannah T. Perkins ◽

Victoria J. Allan ◽

Thomas A. Waigh

Keyword(s):

Endoplasmic Reticulum ◽

Dynamic Behaviour ◽

Persistence Length ◽

Live Cells ◽

Cell Periphery ◽

Active Motion ◽

Subcellular Organelles ◽

Subcellular Organelle ◽

Analysis Workflow ◽

Network Organisation

AbstractThe endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed an analysis workflow for dynamics of established tubules in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active dynamics were observed. Clear differences in dynamic behaviour were observed for established tubules at different positions within the cell using itemset mining. We found that tubules with activity-driven fluctuations were more likely to be located away from the cell periphery and a population of peripheral tubules with no signs of active motion was found.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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Sigma 1 Receptor, Cholesterol and Endoplasmic Reticulum Contact Sites

Contact ◽

10.1177/25152564211026505 ◽

2021 ◽

Vol 4 ◽

pp. 251525642110265

Author(s):

Vladimir Zhemkov ◽

Jen Liou ◽

Ilya Bezprozvanny

Keyword(s):

Endoplasmic Reticulum ◽

Protein Composition ◽

Sigma 1 Receptor ◽

Functional Roles ◽

Membrane Contact Sites ◽

Contact Sites ◽

Sigma 1 ◽

Membrane Contact ◽

Organelle Communication ◽

Cellular Organelles

Recent studies indicated potential importance of membrane contact sites (MCS) between the endoplasmic reticulum (ER) and other cellular organelles. These MCS have unique protein and lipid composition and serve as hubs for inter-organelle communication and signaling. Despite extensive investigation of MCS protein composition and functional roles, little is known about the process of MCS formation. In this perspective, we propose a hypothesis that MCS are formed not as a result of random interactions between membranes of ER and other organelles but on the basis of pre-existing cholesterol-enriched ER microdomains.

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An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽

10.1177/2515256418814621 ◽

2018 ◽

Vol 1 ◽

pp. 251525641881462

Author(s):

Samantha K. Dziurdzik ◽

Björn D.M. Bean ◽

Elizabeth Conibear

Keyword(s):

Endoplasmic Reticulum ◽

Recent Work ◽

Protein Sorting ◽

Repeat Region ◽

Vacuolar Protein Sorting ◽

Membrane Contact Sites ◽

Contact Sites ◽

Prospore Membrane ◽

Membrane Contact ◽

Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

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Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.789959 ◽

2021 ◽

Vol 9 ◽

Author(s):

Sara Benhammouda ◽

Anjali Vishwakarma ◽

Priya Gatti ◽

Marc Germain

Keyword(s):

Endoplasmic Reticulum ◽

Fluorescence Probes ◽

Proximity Ligation Assay ◽

Proximity Ligation ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Endogenous Proteins ◽

Underlying Mechanisms

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.

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Cellular senescence links mitochondria-ER contacts and aging

Communications Biology ◽

10.1038/s42003-021-02840-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Dorian V. Ziegler ◽

Nadine Martin ◽

David Bernard

Keyword(s):

Endoplasmic Reticulum ◽

Cellular Senescence ◽

Cellular Mechanisms ◽

Membrane Contact Sites ◽

Contact Sites ◽

Recent Advances ◽

Key Players ◽

Membrane Contact

AbstractMembrane contact sites emerged in the last decade as key players in the integration, regulation and transmission of many signals within cells, with critical impact in multiple pathophysiological contexts. Numerous studies accordingly point to a role for mitochondria-endoplasmic reticulum contacts (MERCs) in modulating aging. Nonetheless, the driving cellular mechanisms behind this role remain unclear. Recent evidence unravelled that MERCs regulate cellular senescence, a state of permanent proliferation arrest associated with a pro-inflammatory secretome, which could mediate MERC impact on aging. Here we discuss this idea in light of recent advances supporting an interplay between MERCs, cellular senescence and aging.

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STARD 3 mediates endoplasmic reticulum‐to‐endosome cholesterol transport at membrane contact sites

The EMBO Journal ◽

10.15252/embj.201695917 ◽

2017 ◽

Vol 36 (10) ◽

pp. 1412-1433 ◽

Cited By ~ 92

Author(s):

Léa P Wilhelm ◽

Corinne Wendling ◽

Benoît Védie ◽

Toshihide Kobayashi ◽

Marie‐Pierre Chenard ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cholesterol Transport ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805757115 ◽

2018 ◽

Vol 115 (31) ◽

pp. E7331-E7340 ◽

Cited By ~ 46

Author(s):

Ben Johnson ◽

Ashley N. Leek ◽

Laura Solé ◽

Emily E. Maverick ◽

Tim P. Levine ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Neuronal Cultures ◽

Physical Relationship ◽

Membrane Contact Sites ◽

Contact Sites ◽

Hek Cells ◽

C Terminus ◽

Membrane Contact

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

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