bims-mecosi Biomed News
on Membrane contact sites
Issue of 2021–10–24
eight papers selected by
Verena Kohler, Stockholm University
  1. Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells.
  2. Balancing life and death: BCL-2 family members at diverse ER-mitochondria contact sites.
  3. Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.
  4. A three-organelle complex made by wrappER contacts with peroxisome and mitochondria responds to liver lipid flux changes.
  5. Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole.
  6. Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases.
  7. A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis.
  8. VDAC Modulation of Cancer Metabolism: Advances and Therapeutic Challenges.
  1. Nat Protoc. 2021 Oct 22.
    Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells.
    Tito Calì, Marisa Brini.
      Membrane contact sites between organelles are essential for maintaining cellular homeostasis, which requires the continuous exchange of signaling molecules, ions, nutrients and lipids. Alterations of different contact sites are associated with a wide spectrum of human diseases. However, visualizing and quantifying these contact sites remains a challenge. This protocol describes the use of split-GFP-based contact site sensors (SPLICS) in microscopy applications for mapping organelle contact sites both in fixed and living cells. SPLICS sensors are engineered to express equimolar amounts of the organelle-targeted nonfluorescent β11 and GFP1-10 portions of the split-GFP protein in a single vector, and are capable of reconstituting fluorescence when two opposing membranes come into proximity. Reconstitution will occur only over the cell volume at defined contact sites resulting in a bright signal that can be detected easily and quantified automatically with specific custom-made plugins. The use of minimal targeting sequences facilitates targeting specificity and membrane coverage, avoiding artifacts due to full-length fusion protein overexpression and, thus, possible perturbations of the cell's physiology. SPLICS sensors engineered to simultaneously detect multiple contact sites within the same cell have been generated by exploiting the ability of the β11 GFP fragment to reconstitute different color-shifted variants of the GFP1-10 fragment. Here, we describe a detailed protocol to set up SPLICS expression in living cells (2-3 d), detection and acquisition (1 d), and automated quantification with custom plugins (1-2 d). We also advise on construct design and characterization for novel organelle contacts.
    DOI:  https://doi.org/10.1038/s41596-021-00614-1
  2. FEBS J. 2021 Oct 20.
    Balancing life and death: BCL-2 family members at diverse ER-mitochondria contact sites.
    Robert E Means, Samuel G Katz.
      The outer mitochondrial membrane is a busy place. One essential activity for cellular survival is the regulation of membrane integrity by the BCL-2 family of proteins. Another critical facet of the outer mitochondrial membrane is its close approximation with the endoplasmic reticulum. These mitochondria associated membranes (MAMs) occupy a significant fraction of the mitochondrial surface and serve as key signaling hubs for multiple cellular processes. Each of these pathways may be considered as forming their own specialized MAM sub-type. Interestingly, like membrane permeabilization, most of these pathways play critical roles in regulating cellular survival and death. Recently, the pro-apoptotic BCL-2 family member BOK, has been found within MAMs where it plays important roles in their structure and function. This has led to a greater appreciation that multiple BCL-2 family proteins, which are known to participate in numerous functions throughout the cell, also have roles within MAMs. In this review we evaluate several MAM subsets, their role in cellular homeostasis and the contribution of BCL-2 family members to their functions.
    DOI:  https://doi.org/10.1111/febs.16241
  3. Biomolecules. 2021 Oct 14. pii: 1511. [Epub ahead of print]11(10):
    Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.
    Anna Signorile, Anna Ferretta, Consiglia Pacelli, Nazzareno Capitanio, Paola Tanzarella, Maria Laura Matrella, Alessio Valletti, Domenico De Rasmo, Tiziana Cocco.
      Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER-mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.
    Keywords:  cAMP; calcium (Ca2+); endoplasmic reticulum (ER); mitochondria; parkin; resveratrol
    DOI:  https://doi.org/10.3390/biom11101511
  4. J Cell Sci. 2021 Oct 21. pii: jcs.259091. [Epub ahead of print]
    A three-organelle complex made by wrappER contacts with peroxisome and mitochondria responds to liver lipid flux changes.
    Nicolò Ilacqua, Irene Anastasia, Andrea Raimondi, Philippe Lemieux, Thomas Q de Aguiar Vallim, Katalin Toth, Eugene V Koonin, Luca Pellegrini.
      Hepatic lipid homeostasis depends on intracellular pathways that respire fatty acid (FA) in peroxisomes and mitochondria and on systemic pathways that secrete FA into the bloodstream, either free or condensed in very-low-density lipoprotein (VLDL) triglycerides. These systemic and intracellular pathways are interdependent, but it is unclear whether and how they integrate into a single cellular circuit. Here, we report that mouse liver wrappER, a distinct ER compartment with apparent FA- and VLDL-secretion functions, connects peroxisomes and mitochondria. Correlative light electron microscopy, quantitative serial section electron tomography, and 3D organelle reconstruction analysis show that the number of peroxisome-wrappER-mitochondria complexes changes throughout fasting-to-feeding transitions and doubles when VLDL synthesis stops following acute genetic ablation of Mttp in the liver. Quantitative proteomic analysis of peroxisome-wrappER-mitochondria complex-enriched fractions indicates that the loss of Mttp upregulates global FA β-oxidation, thereby integrating the dynamics of this three-organelle association into hepatic FA flux responses. Therefore, liver lipid homeostasis occurs through the convergence of systemic and intracellular FA-elimination pathways in the peroxisome-wrappER-mitochondria complex.
    Keywords:  Fatty acid; Inter-organelle contacts; Liver lipid homeostasis; Mitochondria; Peroxisome; WrappER
    DOI:  https://doi.org/10.1242/jcs.259091
  5. Mol Biol Cell. 2021 Oct 20. mbcE21050227
    Subunit exchange among endolysosomal tethering complexes is linked to contact site formation at the vacuole.
    Ayelén González Montoro, Prado Vargas Duarte, Kathrin Auffarth, Stefan Walter, Florian Fröhlich, Christian Ungermann.
      The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.
    DOI:  https://doi.org/10.1091/mbc.E21-05-0227
  6. Cells. 2021 Sep 23. pii: 2518. [Epub ahead of print]10(10):
    Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases.
    Valentina Tedeschi, Daniele La Russa, Cristina Franco, Antonio Vinciguerra, Diana Amantea, Agnese Secondo.
      Located at the level of the endoplasmic reticulum (ER) membrane, stromal interacting molecule 1 (STIM1) undergoes a complex conformational rearrangement after depletion of ER luminal Ca2+. Then, STIM1 translocates into discrete ER-plasma membrane (PM) junctions where it directly interacts with and activates plasma membrane Orai1 channels to refill ER with Ca2+. Furthermore, Ca2+ entry due to Orai1/STIM1 interaction may induce canonical transient receptor potential channel 1 (TRPC1) translocation to the plasma membrane, where it is activated by STIM1. All these events give rise to store-operated calcium entry (SOCE). Besides the main pathway underlying SOCE, which mainly involves Orai1 and TRPC1 activation, STIM1 modulates many other plasma membrane proteins in order to potentiate the influxof Ca2+. Furthermore, it is now clear that STIM1 may inhibit Ca2+ currents mediated by L-type Ca2+ channels. Interestingly, STIM1 also interacts with some intracellular channels and transporters, including nuclear and lysosomal ionic proteins, thus orchestrating organellar Ca2+ homeostasis. STIM1 and its partners/effectors are significantly modulated in diverse acute and chronic neurodegenerative conditions. This highlights the importance of further disclosing their cellular functions as they might represent promising molecular targets for neuroprotection.
    Keywords:  Ca2+ homeostasis; Ca2+-storing organelles; amyotrophic lateral sclerosis (ALS); endoplasmic reticulum (ER); lysosomes
    DOI:  https://doi.org/10.3390/cells10102518
  7. Protein Cell. 2021 Oct 23.
    A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis.
    Tengfei Ma, Liyuan Zhao, Jie Zhang, Ruofeng Tang, Xin Wang, Nan Liu, Qian Zhang, Fengyang Wang, Meijiao Li, Qian Shan, Yang Yang, Qiuyuan Yin, Limei Yang, Qiwen Gan, Chonglin Yang.
      Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions.
    Keywords:  C. elegans; ER-mitochondrial contact; Zn2+ transporter; development; mitochondria
    DOI:  https://doi.org/10.1007/s13238-021-00881-4
  8. Front Physiol. 2021 ;12 742839
    VDAC Modulation of Cancer Metabolism: Advances and Therapeutic Challenges.
    Kareem A Heslop, Veronica Milesi, Eduardo N Maldonado.
      Most anionic metabolites including respiratory substrates, glycolytic adenosine triphosphate (ATP), and small cations that enter mitochondria, and mitochondrial ATP moving to the cytosol, cross the outer mitochondrial membrane (OMM) through voltage dependent anion channels (VDAC). The closed states of VDAC block the passage of anionic metabolites, and increase the flux of small cations, including calcium. Consequently, physiological or pharmacological regulation of VDAC opening, by conditioning the magnitude of both anion and cation fluxes, is a major contributor to mitochondrial metabolism. Tumor cells display a pro-proliferative Warburg phenotype characterized by enhanced aerobic glycolysis in the presence of partial suppression of mitochondrial metabolism. The heterogeneous and flexible metabolic traits of most human tumors render cells able to adapt to the constantly changing energetic and biosynthetic demands by switching between predominantly glycolytic or oxidative phenotypes. Here, we describe the biological consequences of changes in the conformational state of VDAC for cancer metabolism, the mechanisms by which VDAC-openers promote cancer cell death, and the advantages of VDAC opening as a valuable pharmacological target. Particular emphasis is given to the endogenous regulation of VDAC by free tubulin and the effects of VDAC-tubulin antagonists in cancer cells. Because of its function and location, VDAC operates as a switch to turn-off mitochondrial metabolism (closed state) and increase aerobic glycolysis (pro-Warburg), or to turn-on mitochondrial metabolism (open state) and decrease glycolysis (anti-Warburg). A better understanding of the role of VDAC regulation in tumor progression is relevant both for cancer biology and for developing novel cancer chemotherapies.
    Keywords:  Warburg; cancer; glycolysis; metabolic flexibility; metabolic reprogramming; metabolism; mitochondria; voltage dependent anion channels
    DOI:  https://doi.org/10.3389/fphys.2021.742839
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