Local PI(4,5)P2 pool dynamics detected by the coincidence biosensor tubbyCT maintain the integrity of ER-PM junctions during PLC signaling

ABSTRACTPhosphoinositides (PIs) are important signaling molecules and determinants of membrane identity in the eukaryotic plasma membrane, where they multi-task in divergent signaling pathways. Signaling pleiotropy likely depends on distinct PI pools in the same membrane, although the physical definition of such pools has remained ambiguous. PI(4,5)P2, specifically, is also the precursor for the second messengers in the Gq/PLC pathway, IP3 and DAG, and is broken down by PLCβ during signaling. Endoplasmic reticulum-plasma membrane contact sites (ER-PM junctions) have emerged as central hubs for lipid transport between both membranes, and specifically for PI homeostasis by supplying the PM with phosphatidylinositol.Here we show that the tubby protein, by virtue of its C-terminal tubby-domain, preferentially localizes to ER-PM junctions by binding to both PI(4,5)P2 and the ER-PM tether E-Syt3. Under conditions of vigorous PI(4,5)P2 consumption by PLCβ, additional recruitment of tubby revealed an increase of a local PI(4,5)P2 pool fed by local synthesis through PI kinases. Inhibition of this pool-filling process led to the release of the ER-PM tethers, E-Syts, from the membrane and hence to loss of integrity of the ER-PM contact sites.We conclude that spatiotemporal metabolic channeling of PI synthesis initiated by non-vesicular transport in the ER-PM junctions specifies a local pool of PI(4,5)P2 that is pivotal for the maintenance of homeostatic functions during global depletion of PI(4,5)P2. The findings further suggest that the tubby-like proteins (TULPs), so far known to impact on energy homeostasis and obesity through primary cilia signaling, have an additional function at ER-PM junctions.HIGHLIGHTSThe tubby domain preferentially assembles into ER-PM junctions due to coincidence detection of PI(4,5)P2 and E-Syt3Tubby recruitment reveals an increase of a local pool of PI(4,5)P2 in ER-PM junctions during PLCβ signalingJunctional PI(4,5)P2 dynamics require local synthesis of PI(4,5)P2Local PI(4,5)P2 supply is required for integrity of ER-PM junctions during PLCβ activity.

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Faculty Opinions recommendation of Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8941961.9505055 ◽

2011 ◽

Author(s):

Davis Ng ◽

Guillaume Thibault

Keyword(s):

Plasma Membrane ◽

Phosphoinositide Metabolism ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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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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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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Synaptotagmin-Associated Endoplasmic Reticulum-Plasma Membrane Contact Sites Are Localized to Immobile ER Tubules

PLANT PHYSIOLOGY ◽

10.1104/pp.18.00498 ◽

2018 ◽

Vol 178 (2) ◽

pp. 641-653 ◽

Cited By ~ 9

Author(s):

Kazuya Ishikawa ◽

Kentaro Tamura ◽

Haruko Ueda ◽

Yoko Ito ◽

Akihiko Nakano ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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Ca2+ and lipid signals hold hands at endoplasmic reticulum-plasma membrane contact sites

The Journal of Physiology ◽

10.1113/jp274957 ◽

2018 ◽

Vol 596 (14) ◽

pp. 2709-2716 ◽

Cited By ~ 21

Author(s):

Tamas Balla

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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PERK and filamin A in actin cytoskeleton remodeling at ER-plasma membrane contact sites

Molecular & Cellular Oncology ◽

10.1080/23723556.2017.1340105 ◽

2017 ◽

Vol 4 (5) ◽

pp. e1340105 ◽

Cited By ~ 3

Author(s):

Alexander R. van Vliet ◽

Patrizia Agostinis

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Filamin A ◽

Membrane Contact Sites ◽

Contact Sites ◽

Cytoskeleton Remodeling ◽

Membrane Contact

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Mitochondria–organelle contact sites: the plot thickens

Biochemical Society Transactions ◽

10.1042/bst20160130 ◽

2017 ◽

Vol 45 (2) ◽

pp. 477-488 ◽

Cited By ~ 16

Author(s):

Yael Elbaz-Alon

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cellular Metabolism ◽

Close Apposition ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Cellular Organelles

Membrane contact sites (MCSs) are areas of close apposition between the membranes of two different organelles that enable non-vesicular transfer of ions and lipids. Recent studies reveal that mitochondria maintain contact sites with organelles other than the endoplasmic reticulum such as the vacuole, plasma membrane and peroxisomes. This review focuses on novel findings achieved mainly in yeast regarding tethers, function and regulation of mitochondria–organelle contact sites. The emerging network of MCSs linking virtually all cellular organelles is highly dynamic and integrated with cellular metabolism.

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Synaptotagmins Maintain Diacylglycerol Homeostasis at Endoplasmic Reticulum-Plasma Membrane Contact Sites during Abiotic Stress

10.1101/2020.07.28.222919 ◽

2020 ◽

Cited By ~ 1

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 ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Phosphatidylinositol 4 ◽

Polar Glycerolipids

SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis 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 WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Download Full-text