bims-mecosi 2024-12-22 papers

bims-mecosi

Biomed News

on Membrane contact sites

Issue of 2024–12–22
thirteen papers selected by
Verena Kohler, Umeå University

ER-LD Membrane Contact Sites: A Budding Area in the Pathogen Survival Strategy.
Measurement of ORP10-Mediated Lipid Countertransport at ER-Endosome Membrane Contact Sites via a Chemically Induced Dimerization Strategy.
Measurement of Lipid Transport in Mitochondria by the MTL Complex.
Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.
Analysis of Phosphatidylinositol Transfer at ER-PM Contact Sites in Receptor-Stimulated Live Cells.
Quercetin preserves mitochondria-endoplasmic reticulum contact sites improving mitochondrial dynamics in aged myocardial cells.
Distribution of the p66Shc Adaptor Protein Among Mitochondrial and Mitochondria-Associated Membranes Fractions in Normal and Oxidative Stress Conditions.
DNA-Assisted Assays for Studying Lipid Transfer Between Membranes.
Galactocerebroside Lipid Nanotubes, a Model Membrane System for Studying Membrane-Associated Proteins on a Molecular Scale.
[New Split-GFP systems to follow organelle contact sites in yeast and human cells].
Ezetimibe Enhances Lipid Droplet and Mitochondria Contact Formation, Improving Fatty Acid Transfer and Reducing Lipotoxicity in Alport Syndrome Podocytes.
Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.
Dysregulation of PI4P in the trans Golgi regions activates the mammalian Golgi stress response.

Contact (Thousand Oaks). 2024 Jan-Dec;7:7 25152564241304196

ER-LD Membrane Contact Sites: A Budding Area in the Pathogen Survival Strategy.

Rajendra Kumar Angara, Margaret F Sladek, Stacey D Gilk.

  The endoplasmic reticulum (ER) and lipid droplets (LDs) are essential organelles involved in lipid synthesis, storage, and transport. Physical membrane contacts between the ER and LDs facilitate lipid and protein exchange and thus play a critical role in regulating cellular lipid homeostasis. Recent research has revealed that ER-LD membrane contact sites are targeted by pathogens seeking to exploit host lipid metabolic processes. Both viruses and bacteria manipulate ER-LD membrane contact sites to enhance their replication and survival within the host. This review discusses the research advancements elucidating the mechanisms by which pathogens manipulate the ER-LD contacts through protein molecular mimicry and host cell protein manipulation, thereby hijacking host lipid metabolic processes to facilitate pathogenesis. Understanding the crosstalk between ER and LDs during infection provides deeper insight into host lipid regulation and uncovers potential therapeutic targets for treating infectious diseases.

Keywords:  endoplasmic reticulum; intracellular pathogens; lipid droplet; membrane contact sites; tether proteins

DOI:  https://doi.org/10.1177/25152564241304196
Methods Mol Biol. 2025 ;2888 13-22

Measurement of ORP10-Mediated Lipid Countertransport at ER-Endosome Membrane Contact Sites via a Chemically Induced Dimerization Strategy.

Asami Kawasaki, Fubito Nakatsu.

  Oxysterol-binding protein (OSBP)-related proteins (ORPs) are a large family of lipid transfer proteins (LTPs) in mammals. ORPs mediate the countertransport of two distinct lipids at membrane contact sites (MCSs). ORP10 is localized via binding to ORP9 at the endoplasmic reticulum (ER)-endosome MCSs, where it mediates countertransport of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine (PS). To quantitatively monitor the lipid countertransport process mediated by ORP10 in situ, we take advantage of chemically induced dimerization (CID), a strategy of inducing protein-protein interactions by exposure to chemicals. Specifically, we exploit the rapamycin-inducible heterodimerization of FKBP/FRB to acutely recruit the lipid transfer domain of ORP10 to the ER-endosome MCSs and monitor the levels of PI4P and PS on endosomes by their genetic probes in live imaging. This approach enables the measurement of ORP10 activity in lipid countertransport at ER-endosome MCSs and is also beneficial as a versatile method applicable to other LTPs.

Keywords:  Chemically induced dimerization; Lipid countertransport; Membrane contact sites; Oxysterol-binding protein (OSBP)-related proteins; Phosphatidylinositol 4-phosphate

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_2
Methods Mol Biol. 2025 ;2888 167-191

Measurement of Lipid Transport in Mitochondria by the MTL Complex.

Juliette Jouhet, Valérie Gros, Morgane Michaud.

  Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, less is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria is specifically promoted during phosphate starvation. This lipid exchange likely occurs by non-vesicular trafficking pathways at MCSs between mitochondria and plastids. By a biochemical approach, a mitochondrial lipoproteic super-complex called MTL (mitochondrial transmembrane lipoprotein complex) involved in mitochondrial lipid trafficking has been identified in Arabidopsis thaliana. This protocol describes the method used to separate the MTL complex and to study the implication of a component of this complex (AtMic60) in mitochondrial lipid transport.

Keywords:  CN-PAGE; Lipid transfer; MTL complex; Mass spectrometry; Mitochondria

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_12
Methods Mol Biol. 2025 ;2888 193-200

Determining ATG2 Localization During Autophagosome Formation in Mammalian Cells.

Shenliang Yu.

  This chapter describes two imaging-based approaches for examining the localization of bridge-like lipid transfer proteins at membrane contact sites during native biological processes. These approaches use multi-color fluorescence imaging, enabling high spatial and temporal resolution and overcoming the limitations of biochemical methods. The first approach involves immunofluorescence in fixed cells, while the second utilizes time-lapse imaging in live cells. These methods are showcased through the example of ATG2, an essential autophagy-related protein, and demonstrate the ability to overcome technical difficulties such as large protein size, lack of high-quality antibodies, and imaging highly dynamic subcellular structures. These described methods provide a powerful tool for understanding protein function and biological processes and can be widely applied to various research questions in cell biology.

Keywords:  ATG2; Autophagy; Fluorescence microscopy; Lipid transport; Membrane contact site

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_13
Methods Mol Biol. 2025 ;2888 23-34

Analysis of Phosphatidylinositol Transfer at ER-PM Contact Sites in Receptor-Stimulated Live Cells.

Chi-Lun Chang, Wan-Ru Lee, Wei-Ting Li, Jen Liou.

  Phosphatidylinositol (PI) is an inositol-containing phospholipid synthesized in the endoplasmic reticulum (ER). PI is a precursor lipid for PI 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM) important for Ca2+ signaling in response to extracellular stimuli. Thus, ER-to-PM PI transfer becomes essential for cells to maintain PI(4,5)P2 homeostasis during receptor stimulation. In this chapter, we discuss two live-cell imaging protocols to analyze ER-to-PM PI transfer at ER-PM contact sites, where the two membrane compartments make close appositions accommodating PI transfer. First, we describe how to monitor PI(4,5)P2 replenishment following receptor stimulation as a readout of PI transfer using a PI(4,5)P2 biosensor and total internal reflection microscopy. The second protocol directly visualizes PI transfer proteins that accumulate at ER-PM contact sites and mediate PI(4,5)P2 replenishment with PI in the ER in stimulated cells. These methods provide spatial and temporal analysis of ER-to-PM PI transfer during receptor stimulation and can be adapted to other research questions related to this topic.

Keywords:  ER-PM contact sites; PI; PI transfer protein; PI(4,5)P2 biosensor; PI(4,5)P2 replenishment

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_3
Biogerontology. 2024 Dec 20. 26(1): 29

Quercetin preserves mitochondria-endoplasmic reticulum contact sites improving mitochondrial dynamics in aged myocardial cells.

Ray Jiménez, Alejandra Zúñiga-Muñoz, Edith Álvarez-León, Wylly Ramsés García-Niño, Gabriela Navarrete-Anastasio, Elizabeth Soria-Castro, Israel Pérez-Torres, Elizabeth Lira-Silva, Natalia Pavón, Alfredo Cruz-Gregorio, Rebeca López-Marure, Cecilia Zazueta, Alejandro Silva-Palacios.

  Cardiomyocyte senescence plays a crucial role in the pathophysiology of age-related cardiovascular disease. Senescent cells with impaired contractility, mitochondrial dysfunction, and hypertrophic growth accumulate in the heart during aging, contributing to cardiac dysfunction and remodeling. Mitochondrial dynamics is altered in aging cells, leading to changes in their function and morphology. Such rearrangements can affect the spatially restricted region of the mitochondrial membrane that interacts with reticulum membrane fragments, termed mitochondria-endoplasmic reticulum (ER) contact sites (MERCs). Besides, oxidative stress associated with inefficient organelle turnover can drive cellular senescence. Therefore, in this study, we evaluated the possible association between the senolytic effect of the antioxidant quercetin (Q) and MERCs preservation in a D-galactose-induced cellular senescence model. We found that Q ameliorates the senescent phenotype of H9c2 cells in association with increased mitochondria-ER colocalization, reduced distance between both organelles, and lower ROS production. Moreover, regulation of fusion and fission processes was related with increased mitochondrial ATP production and enhanced transmembrane potential. Overall, our data provide evidence that the inhibitory effect of Q on cellular senescence is associated with preserved MERCs and improved mitochondrial function and morphology, which might contribute to the attenuation of cardiac dysfunction.

Keywords:  Cardiac senescence; Mitochondria-endoplasmic reticulum contact sites; Mitochondrial dynamic; Quercetin; Senolysis

DOI:  https://doi.org/10.1007/s10522-024-10174-y
Int J Mol Sci. 2024 Nov 29. pii: 12835. [Epub ahead of print]25(23):

Distribution of the p66Shc Adaptor Protein Among Mitochondrial and Mitochondria-Associated Membranes Fractions in Normal and Oxidative Stress Conditions.

Magdalena Lebiedzinska-Arciszewska, Barbara Pakula, Massimo Bonora, Sonia Missiroli, Yaiza Potes, Patrycja Jakubek-Olszewska, Ines C M Simoes, Paolo Pinton, Mariusz R Wieckowski.

  p66Shc is an adaptor protein and one of the cellular fate regulators since it modulates mitogenic signaling pathways, mitochondrial function, and reactive oxygen species (ROS) production. p66Shc is localized mostly in the cytosol and endoplasmic reticulum (ER); however, under oxidative stress, p66Shc is post-translationally modified and relocates to mitochondria. p66Shc was found in the intermembrane space, where it interacts with cytochrome c, contributing to the hydrogen peroxide generation by the mitochondrial respiratory chain. Our previous studies suggested that p66Shc is localized also in mitochondria-associated membranes (MAM). MAM fraction consists of mitochondria and mostly ER membranes. Contact sites between ER and mitochondria host proteins involved in multiple processes including calcium homeostasis, apoptosis, and autophagy regulation. Thus, p66Shc in MAM could participate in processes related to cell fate determination. Due to reports on various and conditional p66Shc intracellular localization, in the present paper, we describe the allocation of p66Shc pools in different subcellular compartments in mouse liver tissue and HepG2 cell culture. We provide additional evidence for p66Shc localization in MAM. In the present study, we use precisely purified subcellular fraction isolated by differential centrifugation-based protocol from control mouse liver tissue and HepG2 cells and from cells treated with hydrogen peroxide to promote mitochondrial p66Shc translocation. We performed controlled digestion of crude mitochondrial fraction, in which the degradation patterns of p66Shc and MAM fraction marker proteins were comparable. Moreover, we assessed the distribution of the individual ShcA isoforms (p46Shc, p52Shc, and p66Shc) in the subcellular fractions and their contribution to the total ShcA in control mice livers and HepG2 cells. In conclusion, we showed that a substantial pool of p66Shc protein resides in MAM in control conditions and after oxidative stress induction.

Keywords:  fractionation; mitochondria; mitochondria—ER contact sites; oxidative stress

DOI:  https://doi.org/10.3390/ijms252312835
Methods Mol Biol. 2025 ;2888 221-236

DNA-Assisted Assays for Studying Lipid Transfer Between Membranes.

Yunyun Wang, Qian Shi, Qiulan Yang, Yang Yang, Xin Bian.

  Extended-synaptotagmins (E-Syts) are proteins located on the endoplasmic reticulum (ER) that tether the ER to the plasma membrane (PM) and regulate their lipid homeostasis via its lipid transfer module, the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain. Here, we describe in vitro DNA nanostructure-assisted lipid transfer assays investigating how the SMP domain transports lipids between membranes and associates with the membranes to extract and release lipids. The lipid transfer signal was detected through fluorescence resonance energy transfer (FRET). This method overcomes the limitations of commonly used lipid transfer assays in accurately controlling inter-liposome distance and liposome size, enabling us to further understand the details involved in the process of SMP domain-mediated lipid transfer. Similar platforms can be extended to studying the lipid transfer distance and membrane curvature sensitivity of other lipid transfer proteins.

Keywords:  DNA nanostructure; E-Syts; Membrane contact sites; SMP domain

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_15
Methods Mol Biol. 2025 ;2888 237-248

Galactocerebroside Lipid Nanotubes, a Model Membrane System for Studying Membrane-Associated Proteins on a Molecular Scale.

Aurélie Di Cicco, John Manzi, Julien Maufront, Xingyi Cheng, Manuela Dezi, Daniel Lévy.

  Galactocerebroside lipid nanotubes are membrane-mimicking systems for studying the function and structure of proteins involved in membrane shape remodeling, such as in intracellular trafficking, cell division, and migration or involved in the formation of membrane contact sites. They exhibit a constant and small diameter of 30 nm and a length of up to 2 μm. They can be functionalized with lipid ligands, providing a large binding surface for protein without membrane shape remodeling. These features make it possible to study protein assemblies on membranes different from those accessible with vesicular systems. This chapter describes the process of galactocerebroside nanotube formation, the incorporation of different lipid ligands, factors influencing protein binding, and the experimental conditions for their use in flotation assay and imaging by transmission electron and cryo-electron microscopy.

Keywords:  Biogenesis of lysosome-related organelle complex 1 (BLOC-1); Cryo-electron microscopy; Galactocerebroside; Lipid nanotube; Membrane model; Membrane-bound protein; OSBP; VAP-A

DOI:  https://doi.org/10.1007/978-1-0716-4318-1_16
Med Sci (Paris). 2024 Nov;40(11): 873-875

[New Split-GFP systems to follow organelle contact sites in yeast and human cells].

Carine Amseyan, Amy Blondeau, Julien Karazi, Marie Erard.

DOI: https://doi.org/10.1051/medsci/2024155
Int J Mol Sci. 2024 Dec 06. pii: 13134. [Epub ahead of print]25(23):

Ezetimibe Enhances Lipid Droplet and Mitochondria Contact Formation, Improving Fatty Acid Transfer and Reducing Lipotoxicity in Alport Syndrome Podocytes.

Jin-Ju Kim, Eun-Jeong Yang, Judith Molina David, Sunjoo Cho, Maria Ficarella, Nils Pape, Josephin Elizabeth Schiffer, Rachel Njeim, Stephanie S Kim, Claudia Lo Re, Antonio Fontanella, Maria Kaber, Alexis Sloan, Sandra Merscher, Alessia Fornoni.

  Mitochondrial dysfunction is a critical factor in the pathogenesis of Alport syndrome (AS), contributing to podocyte injury and disease progression. Ezetimibe, a lipid-lowering drug, is known to inhibit cholesterol and fatty acid uptake and to reduce triglyceride content in the kidney cortex of mice with AS. However, its effects on lipid droplet (LD) utilization by mitochondria have not been explored. Transmission electron microscopy (TEM) and mitochondrial functional assays (ATP production, mitochondrial membrane potential, and citrate synthase activity) were used to investigate the impact of ezetimibe on LD-mitochondria contact formation and mitochondrial function in Col4a3KO (AS) and wildtype (WT) podocytes. TEM analysis revealed significant mitochondrial abnormalities in AS podocytes, including swollen mitochondria and reduced cristae density, while mitochondrial function assays showed decreased ATP production and lowered mitochondrial membrane potential. AS podocytes also demonstrated a higher content of LD but with reduced LD-mitochondria contact sites. Ezetimibe treatment significantly increased the number of LD-mitochondria contact sites, enhanced fatty acid transfer efficiency, and reduced intracellular lipid accumulation. These changes were associated with a marked reduction in the markers of lipotoxicity, such as apoptosis and oxidative stress. Mitochondrial function was significantly improved, evidenced by increased basal respiration, ATP production, maximal respiration capacity, and the restoration of mitochondrial membrane potential. Additionally, mitochondrial swelling was significantly reduced in ezetimibe-treated AS podocytes. Our findings reveal a novel role for ezetimibe in enhancing LD-mitochondria contact formation, leading to more efficient fatty acid transfer, reduced lipotoxicity, and improved mitochondrial function in AS podocytes. These results suggest that ezetimibe could be a promising therapeutic agent for treating mitochondrial dysfunction and lipid metabolism abnormalities in AS.

Keywords:  Alport syndrome podocytes; fatty acid; lipid droplet; lipotoxicity

DOI:  https://doi.org/10.3390/ijms252313134
Autophagy. 2024 Dec 15.

Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.

Isabel Gordaliza-Alaguero, Paula Sànchez-Fernàndez-de-Landa, Dragana Radivojevikj, Laura Villarreal, Gianluca Arauz-Garofalo, Marina Gay, Marta Martinez-Vicente, Jorge Seco, Pau Martin-Malpartida, Marta Vilaseca, María J Macías, Manuel Palacin, Saška Ivanova, Antonio Zorzano.

  MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.

Keywords:  Autophagosomes; endosomes; mitochondria; mitochondria-endoplasmic reticulum contact sites; mitochondrial dynamics; nutrient deprivation

DOI:  https://doi.org/10.1080/15548627.2024.2440843
J Biol Chem. 2024 Dec 13. pii: S0021-9258(24)02577-8. [Epub ahead of print] 108075

Dysregulation of PI4P in the trans Golgi regions activates the mammalian Golgi stress response.

Kanae Sasaki, Marika Toide, Takuya Adachi, Fumi Morishita, Yuto Watanabe, Hajime Tajima Sakurai, Sadao Wakabayashi, Satoshi Kusumi, Toshiyuki Yamaji, Kaori Sakurai, Daisuke Koga, Kentaro Hanada, Masafumi Yohda, Hiderou Yoshida.

  The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death. OSW-1, a natural compound with anticancer activity, potently inhibits OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the PI4P pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed by the loss of regulators of PI4P synthesis, such as PITPNB and PI4KB. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi.

Keywords:  Golgi stress response; OSW-1; cancer; phosphatidylinositol-4-phosphate; the Genome-wide CRISPR-Cas9 knockout screening

DOI:  https://doi.org/10.1016/j.jbc.2024.108075