Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles

Science ◽  
2020 ◽  
Vol 367 (6477) ◽  
pp. eaay7108 ◽  
Author(s):  
Jason E. Lee ◽  
Peter I. Cathey ◽  
Haoxi Wu ◽  
Roy Parker ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Granules ◽  
Human Cells ◽  
Stress Granule ◽  
Processing Bodies ◽  
P Bodies ◽  
Contact Sites ◽  
Membrane Bound ◽  
Efficient Transfer ◽  
Er Membrane

Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

scholarly journals Transcriptome-Wide Comparison of Stress Granules and P-Bodies Reveals that Translation Plays a Major Role in RNA Partitioning

2019 ◽  
Vol 39 (24) ◽  
Author(s):  
Tyler Matheny ◽  
Bhalchandra S. Rao ◽  
Roy Parker
Keyword(s):  
Stress Granules ◽  
Stress Granule ◽  
Dominant Factor ◽  
Differential Centrifugation ◽  
P Bodies ◽  
Translation Repression ◽  
First Approximation ◽  
Rnp Granules ◽  
Interpretation Of Results

ABSTRACT The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.

Escape from stress granule sequestration: another way to drug resistance?

2010 ◽  
Vol 38 (6) ◽  
pp. 1537-1542 ◽  
Author(s):  
Ernesto Yagüe ◽  
Selina Raguz
Keyword(s):  
Endoplasmic Reticulum ◽  
Multidrug Resistance ◽  
Stress Granules ◽  
Stress Granule ◽  
Chemotherapeutic Drugs ◽  
P Glycoprotein ◽  
Mdr1 Mrna ◽  
Leukaemic Cells ◽  
Multidrug Resistance 1 Gene ◽  
Polysome Profile

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance and chemotherapy failure in cancer. We have demonstrated that, in leukaemic cells, P-glycoprotein expression is regulated at the translational level. More recently, we have shown that in cells overexpressing P-glycoprotein, MDR1 mRNA does not aggregate into translationally silent stress granules. Importantly, this is not unique for MDR1, since other transcripts encoding transmembrane proteins, and which are thus translated at the endoplasmic reticulum, follow the same pattern. By using a series of chimaeric transcripts, we have demonstrated that transcript localization at the endoplasmic reticulum bypasses the signals dictating stress granule sequestration. Polysome profile analyses and protein synthesis experiments indicate that, upon stress withdrawal, endoplasmic-reticulum-bound transcripts resume translation faster than those at the cytosol, which have been sequestered into stress granules. This may represent a novel mechanism by which drug-resistant cells respond quickly to stress, helping them to survive the cytotoxic effect of chemotherapeutic drugs.

scholarly journals Multiple C2 domain-containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized ER subdomains

2021 ◽  
pp. mbc.E20-09-0590
Author(s):  
Amit S. Joshi ◽  
Joey V. Ragusa ◽  
William A. Prinz ◽  
Sarah Cohen
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplets ◽  
Transmembrane Domain ◽  
Neutral Lipids ◽  
Transmembrane Proteins ◽  
C2 Domain ◽  
General Role ◽  
C2 Domains ◽  
Contact Sites ◽  
Er Membrane

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs co-localize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites. [Media: see text] [Media: see text]

scholarly journals The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

2011 ◽  
Vol 192 (4) ◽  
pp. 583-598 ◽  
Author(s):  
Cornelia Kurischko ◽  
Hong Kyung Kim ◽  
Venkata K. Kuravi ◽  
Juliane Pratzka ◽  
Francis C. Luca
Keyword(s):  
Binding Protein ◽  
Cellular Stress ◽  
Stress Granules ◽  
Polarized Growth ◽  
Cell Integrity ◽  
Processing Bodies ◽  
P Bodies ◽  
Mrna Binding Protein ◽  
Cortical Localization ◽  
Mrna Binding

The mRNA-binding protein Ssd1 is a substrate for the Saccharomyces cerevisiae LATS/NDR orthologue Cbk1, which controls polarized growth, cell separation, and cell integrity. We discovered that most Ssd1 localizes diffusely within the cytoplasm, but some transiently accumulates at sites of polarized growth. Cbk1 inhibition and cellular stress cause Ssd1 to redistribute to mRNA processing bodies (P-bodies) and stress granules, which are known to repress translation. Ssd1 recruitment to P-bodies is independent of mRNA binding and is promoted by the removal of Cbk1 phosphorylation sites. SSD1 deletion severely impairs the asymmetric localization of the Ssd1-associated mRNA, SRL1. Expression of phosphomimetic Ssd1 promotes polarized localization of SRL1 mRNA, whereas phosphorylation-deficient Ssd1 causes constitutive localization of SRL1 mRNA to P-bodies and causes cellular lysis. These data support the model that Cbk1-mediated phosphorylation of Ssd1 promotes the cortical localization of Ssd1–mRNA complexes, whereas Cbk1 inhibition, cellular stress, and Ssd1 dephosphorylation promote Ssd1–mRNA interactions with P-bodies and stress granules, leading to translational repression.

scholarly journals P bodies promote stress granule assembly in Saccharomyces cerevisiae

2008 ◽  
Vol 183 (3) ◽  
pp. 441-455 ◽  
Author(s):  
J. Ross Buchan ◽  
Denise Muhlrad ◽  
Roy Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Protein Composition ◽  
Stress Granules ◽  
Glucose Deprivation ◽  
Stress Granule ◽  
Granule Formation ◽  
P Bodies ◽  
Rna Granules ◽  
Kinetics Of

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

Acidic stress induces the formation of P-bodies, but not stress granules, with mild attenuation of bulk translation in Saccharomyces cerevisiae

2012 ◽  
Vol 446 (2) ◽  
pp. 225-233 ◽  
Author(s):  
Aya Iwaki ◽  
Shingo Izawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Stress Granules ◽  
Glucose Deprivation ◽  
Acidic Stress ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Glucose Depletion ◽  
Messenger Ribonucleoprotein

The stress response of eukaryotic cells often causes an attenuation of bulk translation activity and the accumulation of non-translating mRNAs into cytoplasmic mRNP (messenger ribonucleoprotein) granules termed cytoplasmic P-bodies (processing bodies) and SGs (stress granules). We examined effects of acidic stress on the formation of mRNP granules compared with other forms of stress such as glucose deprivation and a high Ca2+ level in Saccharomyces cerevisiae. Treatment with lactic acid clearly caused the formation of P-bodies, but not SGs, and also caused an attenuation of translation initiation, albeit to a lesser extent than glucose depletion. P-body formation was also induced by hydrochloric acid and sulfuric acid. However, lactic acid in SD (synthetic dextrose) medium with a pH greater than 3.0, propionic acid and acetic acid did not induce P-body formation. The results of the present study suggest that the assembly of yeast P-bodies can be induced by external conditions with a low pH and the threshold was around pH 2.5. The P-body formation upon acidic stress required Scd6 (suppressor of clathrin deficiency 6), a component of P-bodies, indicating that P-bodies induced by acidic stress have rules of assembly different from those induced by glucose deprivation or high Ca2+ levels.

scholarly journals Endoplasmic Reticulum‐Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling

2021 ◽  
Vol 22 (9) ◽  
pp. 4703
Author(s):  
Tim Crul ◽  
József Maléth
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Second Messengers ◽  
Adenosine Monophosphate ◽  
Camp Signaling ◽  
Cell Physiology ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Er Membrane

In eukaryotic cells, ultimate specificity in activation and action—for example, by means of second messengers—of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple—sometimes opposite—cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER–PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

scholarly journals Decapping complex is essential for functional P-body formation and is buffered by nuclear localization

2020 ◽  
Author(s):  
Kiril Tishinov ◽  
Anne Spang
Keyword(s):  
Endoplasmic Reticulum ◽  
Phase Separation ◽  
Nuclear Localization ◽  
Mrna Decay ◽  
Dynamic Equilibrium ◽  
Local Concentration ◽  
Processing Bodies ◽  
Body Formation ◽  
P Bodies ◽  
Translational Repressor

AbstractmRNA decay is a key step in regulating the cellular proteome. Cytoplasmic mRNA is largely turned over in processing bodies (P-bodies). P-body units assemble to form P-body granules under stress conditions. How this assembly is regulated, however, remains still poorly understood. Here, we show that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by capturing the Dcp1/2 decapping complex and preventing it from becoming imported into the nucleus by the karyopherin ß Kap95. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a ready releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3 and Scd6. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Scd6 and Edc3 to drive phase separation and hence P-body formation.

scholarly journals Getting a handle on lipid droplets: Insights into ER–lipid droplet tethering

2019 ◽  
Vol 218 (4) ◽  
pp. 1089-1091 ◽  
Author(s):  
Truc B. Nguyen ◽  
James A. Olzmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Metabolism ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Human Cells ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cell Biol ◽  
Membrane Contact

Lipid droplets (LDs) are hubs for lipid metabolism that form membrane contact sites with multiple organelles. In this issue, Hariri et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808119) reveal the functions of Mdm1-mediated endoplasmic reticulum (ER)–LD tethering in yeast and Datta et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808133) identify a role for the Mdm1 orthologue, Snx14, as an ER–LD tether that regulates lipid metabolism in human cells.

scholarly journals Novel Aspects of Degradation of T Cell Receptor Subunits from the Endoplasmic Reticulum (ER) in T Cells: Importance of Oligosaccharide Processing, Ubiquitination, and Proteasome-dependent Removal from ER Membranes

1998 ◽  
Vol 187 (6) ◽  
pp. 835-846 ◽  
Author(s):  
Mei Yang ◽  
Satoshi Omura ◽  
Juan S. Bonifacino ◽  
Allan M. Weissman
Keyword(s):  
T Cells ◽  
Endoplasmic Reticulum ◽  
T Cell ◽  
Secretory Pathway ◽  
Cell Receptor ◽  
Dependent Manner ◽  
Oligosaccharide Processing ◽  
Membrane Bound ◽  
Α Subunits ◽  
Er Membrane

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as “ER degradation”. The molecular basis for the loss of the TCR CD3-δ and TCR-α subunits from the ER was investigated in lymphocytes. For CD3-δ, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-δ was markedly curtailed and CD3-δ remained membrane bound in a complex with CD3-ε. TCR-α was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-α, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation.

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