scholarly journals Fluorescent Tools to Analyze Peroxisome–Endoplasmic Reticulum Interactions in Mammalian Cells

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641984864 ◽  
Author(s):  
Alexa Bishop ◽  
Maki Kamoshita ◽  
Josiah B. Passmore ◽  
Christian Hacker ◽  
Tina A. Schrader ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Proximity Ligation Assay ◽  
Reporter System ◽  
Peroxisomal Membrane ◽  
Proximity Ligation ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins, ACBD5 and ACBD4, and the ER protein vesicle-associated membrane protein-associated protein-B (VAPB) as tethering components, which physically interact to foster PO–ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of PO–ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into PO–ER associations at the level of membrane contact sites, their role, composition, and regulation, we have developed two fluorescence-based systems to monitor PO–ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay, we were able to measure the changes in PO–ER interactions while the split-fluorescence reporter was more limited and only allowed us to label PO–ER contacts. We show that both techniques can be useful additions to the toolkit of methods to study PO–ER associations and explore the relative merits of each.

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

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.

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

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.

scholarly journals ORP5 Transfers Phosphatidylserine To Mitochondria And Regulates Mitochondrial Calcium Uptake At Endoplasmic Reticulum - Mitochondria Contact Sites

2019 ◽  
Author(s):  
Leila Rochin ◽  
Cécile Sauvanet ◽  
Eeva Jääskeläinen ◽  
Audrey Houcine ◽  
Amita Arora ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Calcium Uptake ◽  
Vesicular Transport ◽  
Mitochondrial Calcium ◽  
Mitochondrial Membranes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Mitochondrial Calcium Uptake

SUMMARYMitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the two mitochondrial membranes. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at sites of close organelle apposition called membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that ORP5/8 mediate non-vesicular transport of Phosphatidylserine (PS) from the ER to mitochondria in mammalian cells. We also show that ER-mitochondria contacts where ORP5/8 reside are physically and functionally linked to the MIB/MICOS complexes that bridge the mitochondrial membranes, cooperating with them to facilitate PS transfer from the ER to the mitochondria. Finally, we show that ORP5 but not ORP8, additionally regulates import of calcium to mitochondria and consequently cell senescence.

scholarly journals OSBP-Related Protein Family in Lipid Transport over Membrane Contact Sites

2015 ◽  
Vol 8s1 ◽  
pp. LPI.S31726 ◽  
Author(s):  
Vesa M. Olkkonen
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Binding Protein ◽  
High Capacity ◽  
Retrograde Transport ◽  
Protein Family ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.

scholarly journals Sigma 1 Receptor, Cholesterol and Endoplasmic Reticulum Contact Sites

Contact ◽  
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.

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

Contact ◽  
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.

scholarly journals Cellular senescence links mitochondria-ER contacts and aging

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.

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

2017 ◽  
Vol 36 (10) ◽  
pp. 1412-1433 ◽  
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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