bims-mecosi Biomed News
on Membrane contact sites
Issue of 2023–11–12
eleven papers selected by
Verena Kohler, Umeå University
  1. Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays.
  2. ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.
  3. ER-mitochondria contact sites in mitochondrial DNA dynamics, maintenance, and distribution.
  4. Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria-ER contacts.
  5. Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.
  6. Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.
  7. Burst of hopping trafficking correlated reversible dynamic interactions between lipid droplets and mitochondria under starvation.
  8. The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions.
  9. Physical Contacts Between Mitochondria and WPBs Participate in WPB Maturation.
  10. Endoplasmic reticulum regulation of glucose metabolism in glioma stem cells.
  11. ARL6IP1 gene delivery reduces neuroinflammation and neurodegenerative pathology in hereditary spastic paraplegia model.
  1. J Vis Exp. 2023 Oct 20.
    Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays.
    Vera Filipa Monteiro-Cardoso, Romain Le Bars, Francesca Giordano.
      Membrane contact sites (MCSs) are areas of close membrane proximity that allow and regulate the dynamic exchange of diverse biomolecules (i.e., calcium and lipids) between the juxtaposed organelles without involving membrane fusion. MCSs are essential for cellular homeostasis, and their functions are ensured by the resident components, which often exist as multimeric protein complexes. MCSs often involve the endoplasmic reticulum (ER), a major site of lipid synthesis and cellular calcium storage, and are particularly important for organelles, such as the mitochondria, which are excluded from the classical vesicular transport pathways. In the last years, MCSs between the ER and mitochondria have been extensively studied, as their functions strongly impact cellular metabolism/bioenergetics. Several proteins have started to be identified at these contact sites, including membrane tethers, calcium channels, and lipid transfer proteins, thus raising the need for new methodologies and technical approaches to study these MCS components. Here, we describe a protocol consisting of combined technical approaches, that include proximity ligation assay (PLA), mitochondria staining, and 3D imaging segmentation, that allows the detection of proteins that are physically close (>40 nm) to each other and that reside on the same membrane at ER-mitochondria MCSs. For instance, we used two ER-anchored lipid transfer proteins, ORP5 and ORP8, which have previously been shown to interact and localize at ER-mitochondria and ER-plasma membrane MCSs. By associating the ORP5-ORP8 PLA with cell imaging software analysis, it was possible to estimate the distance of the ORP5-ORP8 complex from the mitochondrial surface and determine that about 50% of ORP5-ORP8 PLA interaction occurs at ER subdomains in close proximity to mitochondria.
    DOI:  https://doi.org/10.3791/64750
  2. Life Sci Alliance. 2024 Jan;pii: e202302335. [Epub ahead of print]7(1):
    ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.
    Alexandre Janer, Jordan L Morris, Michiel Krols, Hana Antonicka, Mari J Aaltonen, Zhen-Yuan Lin, Hanish Anand, Anne-Claude Gingras, Julien Prudent, Eric A Shoubridge.
      Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1-SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria-ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.
    DOI:  https://doi.org/10.26508/lsa.202302335
  3. Int J Biochem Cell Biol. 2023 Nov 04. pii: S1357-2725(23)00131-0. [Epub ahead of print] 106492
    ER-mitochondria contact sites in mitochondrial DNA dynamics, maintenance, and distribution.
    Hema Saranya Ilamathi, Marc Germain.
      Mitochondria are central cellular metabolic hubs. Their function requires proteins encoded by nuclear DNA, but also mitochondrial DNA (mtDNA) whose maintenance is essential for the proper function of the organelle. Defective mtDNA maintenance and distribution are associated with mitochondrial diseases. mtDNA is organized into nucleo-protein complexes called nucleoids that dynamically move along the mitochondrial network and interact with each other. mtDNA replication and nucleoid distribution is an active process regulated by the complex interplay of mitochondrial dynamics, endoplasmic reticulum (ER)-mitochondria contact sites, and cytoskeletal networks. For example, defects in mitochondrial fusion and fission or ER-mitochondria contact sites affect nucleoid maintenance and distribution. In this review, we discuss the process of nucleoid dynamics and the factors regulating nucleoid maintenance and distribution.
    Keywords:  ER sheets; ERMCS; mitochondria; mtDNA-nucleoid
    DOI:  https://doi.org/10.1016/j.biocel.2023.106492
  4. J Cell Biol. 2024 Jan 01. pii: e202206109. [Epub ahead of print]223(1):
    Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria-ER contacts.
    Ben Cardoen, Kurt R Vandevoorde, Guang Gao, Milene Ortiz-Silva, Parsa Alan, William Liu, Ellie Tiliakou, A Wayne Vogl, Ghassan Hamarneh, Ivan R Nabi.
      Identification and morphological analysis of mitochondria-ER contacts (MERCs) by fluorescent microscopy is limited by subpixel resolution interorganelle distances. Here, the membrane contact site (MCS) detection algorithm, MCS-DETECT, reconstructs subpixel resolution MERCs from 3D super-resolution image volumes. MCS-DETECT shows that elongated ribosome-studded riboMERCs, present in HT-1080 but not COS-7 cells, are morphologically distinct from smaller smooth contacts and larger contacts induced by mitochondria-ER linker expression in COS-7 cells. RiboMERC formation is associated with increased mitochondrial potential, reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 and HeLa cells. Knockdown of riboMERC tether RRBP1 eliminates riboMERCs in both wild-type and Gp78 knockout HT-1080 cells. By MCS-DETECT, Gp78-dependent riboMERCs present complex tubular shapes that intercalate between and contact multiple mitochondria. MCS-DETECT of 3D whole-cell super-resolution image volumes, therefore, identifies novel dual control of tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.
    DOI:  https://doi.org/10.1083/jcb.202206109
  5. Proteomics. 2023 Nov 05. e2300099
    Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.
    Bahnisikha Barman, Marisol Ramirez, Toni Renee Dawson, Qi Liu, Alissa M Weaver.
      Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid, and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein vesicle associated protein associated protein A (VAP-A) drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and upregulated proteins in small EVs (SEVs) purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. Search Tool for the Retrieval of Reciprocity Genes (STRING) protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD SEVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.
    Keywords:  endoplasmic reticulum; extracellular vesicles; gene set enrichment analysis; membrane contact sites; vesicle associated protein-associated protein A (VAP-A)
    DOI:  https://doi.org/10.1002/pmic.202300099
  6. Traffic. 2023 Nov 05.
    Peroxisome population control by phosphoinositide signaling at the endoplasmic reticulum-plasma membrane interface.
    Barbara Knoblach, Richard A Rachubinski.
      Phosphoinositides are lipid signaling molecules acting at the interface of membranes and the cytosol to regulate membrane trafficking, lipid transport and responses to extracellular stimuli. Peroxisomes are multicopy organelles that are highly responsive to changes in metabolic and environmental conditions. In yeast, peroxisomes are tethered to the cell cortex at defined focal structures containing the peroxisome inheritance protein, Inp1p. We investigated the potential impact of changes in cortical phosphoinositide levels on the peroxisome compartment of the yeast cell. Here we show that the phosphoinositide, phosphatidylinositol-4-phosphate (PI4P), found at the junction of the cortical endoplasmic reticulum and plasma membrane (cER-PM) acts to regulate the cell's peroxisome population. In cells lacking a cER-PM tether or the enzymatic activity of the lipid phosphatase Sac1p, cortical PI4P is elevated, peroxisome numbers and motility are increased, and peroxisomes are no longer firmly tethered to Inp1p-containing foci. Reattachment of the cER to the PM through an artificial ER-PM "staple" in cells lacking the cER-PM tether does not restore peroxisome populations to the wild-type condition, demonstrating that integrity of PI4P signaling at the cell cortex is required for peroxisome homeostasis.
    Keywords:  Inp1p; PI4P; Sac1p; endoplasmic reticulum; lipid phosphatase; membrane contact site; peroxisome; phosphoinositide; plasma membrane; yeast
    DOI:  https://doi.org/10.1111/tra.12923
  7. Exploration (Beijing). 2023 Oct;3(5): 20230002
    Burst of hopping trafficking correlated reversible dynamic interactions between lipid droplets and mitochondria under starvation.
    Zhongju Ye, Chengyuan Hu, Junli Wang, Hua Liu, Luping Li, Jie Yuan, Ji Won Ha, Zhaohui Li, Lehui Xiao.
      Dynamic membrane contacts between lipid droplets (LDs) and mitochondria play key roles in lipid metabolism and energy homeostasis. Understanding the dynamics of LDs under energy stimulation is thereby crucial to disclosing the metabolic mechanism. Here, the reversible interactions between LDs and mitochondria are tracked in real-time using a robust LDs-specific fluorescent probe (LDs-Tags). Through tracking the dynamics of LDs at the single-particle level, spatiotemporal heterogeneity is revealed. LDs in starved cells communicate and integrate their activities (i.e., lipid exchange) through a membrane contact site-mediated mechanism. Thus the diffusion is intermittently alternated between active and confined states. Statistical analysis shows that the translocation of LDs in response to starvation stress is non-Gaussian, and obeys nonergodic-like behavior. These results provide deep understanding of the anomalous diffusion of LDs in living cells, and also afford guidance for rationally designing efficient transporter.
    Keywords:  dynamic contact; fluorescence imaging; lipid droplet; mitochondrion; single‐particle tracking
    DOI:  https://doi.org/10.1002/EXP.20230002
  8. Cells. 2023 Oct 26. pii: 2527. [Epub ahead of print]12(21):
    The Complex Effects of PKM2 and PKM2:IP3R Disruption on Intracellular Ca2+ Handling and Cellular Functions.
    Fernanda O Lemos, Ian de Ridder, Martin D Bootman, Geert Bultynck, Jan B Parys.
      Pyruvate kinase M (PKM) 2 was described to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and suppress its activity. To further investigate the physiological importance of the PKM2:IP3R interaction, we developed and characterized HeLa PKM2 knockout (KO) cells. In the HeLa PKM2 KO cells, the release of Ca2+ to the cytosol appears to be more sensitive to low agonist concentrations than in HeLa wild-type (WT) cells. However, upon an identical IP3-induced Ca2+ release, Ca2+ uptake in the mitochondria is decreased in HeLa PKM2 KO cells, which may be explained by the smaller number of contact sites between the ER and the mitochondria. Furthermore, in HeLa PKM2 KO cells, mitochondria are more numerous, though they are smaller and less branched and have a hyperpolarized membrane potential. TAT-D5SD, a cell-permeable peptide representing a sequence derived from IP3R1 that can disrupt the PKM2:IP3R interaction, induces Ca2+ release into the cytosol and Ca2+ uptake into mitochondria in both HeLa WT and PKM2 KO cells. Moreover, TAT-D5SD induced apoptosis in HeLa WT and PKM2 KO cells but not in HeLa cells completely devoid of IP3Rs. These results indicate that PKM2 separately regulates cytosolic and mitochondrial Ca2+ handling and that the cytotoxic effect of TAT-D5SD depends on IP3R activity but not on PKM2. However, the tyrosine kinase Lck, which also interacts with the D5SD sequence, is expressed neither in HeLa WT nor PKM2 KO cells, and we can also exclude a role for PKM1, which is upregulated in HeLa PKM2 KO cells, indicating that the TAT-D5SD peptide has a more complex mode of action than anticipated.
    Keywords:  ER-mitochondria contact sites; IP3 receptor; apoptosis; intracellular Ca2+ signaling; pyruvate kinase M2
    DOI:  https://doi.org/10.3390/cells12212527
  9. Arterioscler Thromb Vasc Biol. 2023 Nov 09.
    Physical Contacts Between Mitochondria and WPBs Participate in WPB Maturation.
    Jing Ma, Zhenhua Hao, Yudong Zhang, Liuju Li, Xiaoshuai Huang, Yu Wang, Liangyi Chen, Ge Yang, Wei Li.
       BACKGROUND: Weibel-Palade bodies (WPBs) are endothelial cell-specific cigar-shaped secretory organelles containing various biologically active molecules. WPBs play crucial roles in thrombosis, hemostasis, angiogenesis, and inflammation. The main content of WPBs is the procoagulant protein vWF (von Willebrand factor). Physical contacts and functional cross talk between mitochondria and other organelles have been demonstrated. Whether an interorganellar connection exists between mitochondria and WPBs is unknown.
    METHODS: We observed physical contacts between mitochondria and WPBs in human umbilical vein endothelial cells by electron microscopy and living cell confocal microscopy. We developed an artificial intelligence-assisted method to quantify the duration and length of organelle contact sites in live cells.
    RESULTS: We found there existed physical contacts between mitochondria and WPBs. Disruption of mitochondrial function affected the morphology of WPBs. Furthermore, we found that Rab3b, a small GTPase on the WPBs, was enriched at the mitochondrion-WPB contact sites. Rab3b deficiency reduced interaction between the two organelles and impaired the maturation of WPBs and vWF multimer secretion.
    CONCLUSIONS: Our results reveal that Rab3b plays a crucial role in mediating the mitochondrion-WPB contacts, and that mitochondrion-WPB coupling is critical for the maturation of WPBs in vascular endothelial cells.
    Keywords:  Weibel-Palade body; endothelial cells; mitochondrion; organelle interaction; von Willebrand factor
    DOI:  https://doi.org/10.1161/ATVBAHA.123.319939
  10. Int J Oncol. 2024 Jan;pii: 1. [Epub ahead of print]64(1):
    Endoplasmic reticulum regulation of glucose metabolism in glioma stem cells.
    María Turos-Cabal, Ana M Sánchez-Sánchez, Noelia Puente-Moncada, Federico Herrera, Jezabel Rodriguez-Blanco, Isaac Antolin, Marco Antonio Alvarez-Vega, Carmen Rodríguez, Vanesa Martín.
      Glioblastoma (GBM) treatment is extremely challenging due to the high complexity of the tumor. It is one of the tumors in which a subpopulation of highly resistant glioma initiating cells (GICs) has been clearly identified. Thus, understanding the differences between GICs and tumor bulk cells is therefore essential to move to less conventional but more efficient approaches. It was found that, unlike their differentiated progeny, GICs survival and maintenance of stem cell properties depend on mitochondrial metabolism. GICs present higher glucose uptake and mitochondrial membrane potential and reduced lactate dehydrogenase activity, being more sensitive to mitochondrial inhibition than their differentiated counterparts. Calcium flux to the mitochondria appears to play an essential role in the maintenance of this distinct metabolic phenotype with a decrease in the expression of voltage‑dependent anionic channel (VDAC) and Grp75, two of the proteins of the IP3R‑Grp75‑VDAC complex that transfers calcium from the endoplasmic reticulum (ER) to the mitochondria. Disruption of ER homeostasis using ER stress inducers or inhibition of ER‑mitochondrial contact sites using the Grp75 inhibitor MKT‑077 resulted in cytotoxicity of GICs and loss of stemness. Moreover, MKT‑077 also potentiates the effect of temozolomide, current treatment for glioblastoma. In summary, the present data indicated that ER‑mitochondrial homeostasis is essential for regulation of GICs glucose metabolism and survival.
    Keywords:  endoplasmic reticulum; glioma initiating cells; metabolic reprograming; mitochondria; mitochondria‑ER contact sites
    DOI:  https://doi.org/10.3892/ijo.2023.5589
  11. J Exp Med. 2024 Jan 01. pii: e20230367. [Epub ahead of print]221(1):
    ARL6IP1 gene delivery reduces neuroinflammation and neurodegenerative pathology in hereditary spastic paraplegia model.
    Jung Hwa Lim, Hyun Mi Kang, Dae Hun Kim, Bohyeon Jeong, Da Yong Lee, Jae-Ran Lee, Jeong Yeob Baek, Hyun-Soo Cho, Mi-Young Son, Dae Soo Kim, Nam-Soon Kim, Cho-Rok Jung.
      ARL6IP1 is implicated in hereditary spastic paraplegia (HSP), but the specific pathogenic mechanism leading to neurodegeneration has not been elucidated. Here, we clarified the molecular mechanism of ARL6IP1 in HSP using in vitro and in vivo models. The Arl6ip1 knockout (KO) mouse model was generated to represent the clinically involved frameshift mutations and mimicked the HSP phenotypes. Notably, in vivo brain histopathological analysis revealed demyelination of the axon and neuroinflammation in the white matter, including the corticospinal tract. In in vitro experiments, ARL6IP1 silencing caused cell death during neuronal differentiation and mitochondrial dysfunction by dysregulated autophagy. ARL6IP1 localized on mitochondria-associated membranes (MAMs) to maintain endoplasmic reticulum and mitochondrial homeostasis via direct interaction with LC3B and BCl2L13. ARL6IP1 played a crucial role in connecting the endoplasmic reticulum and mitochondria as a member of MAMs. ARL6IP1 gene therapy reduced HSP phenotypes and restored pathophysiological changes in the Arl6ip1 KO model. Our results established that ARL6IP1 could be a potential target for HSP gene therapy.
    DOI:  https://doi.org/10.1084/jem.20230367
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