bims-mecosi 2023-03-26 papers

bims-mecosi

Biomed News

on Membrane contact sites

Issue of 2023–03–26
nine papers selected by
Verena Kohler, University of Graz

Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.
The contribution of mitochondria-associated endoplasmic reticulum membranes (MAMs) dysfunction in Alzheimer's disease and the potential countermeasure.
The Endoplasmic Reticulum and Its Contacts: Emerging Roles in Axon Development, Neurotransmission, and Degeneration.
Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.
Assessing Peroxisomal Protein Interaction by Immunoprecipitation.
Zearalenone induces mitochondria-associated endoplasmic reticulum membranes dysfunction in piglet Sertoli cells based on endoplasmic reticulum stress.
Syntaxin17 contributes to obesity cardiomyopathy through promoting mitochondrial Ca2+ overload in a Parkin-MCUb-dependent manner.
Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.
Correlative Light- and Electron Microscopy in Peroxisome Research.

Methods Mol Biol. 2023 ;2643 135-148

Proximity-Ligation Assay to Detect Peroxisome-Organelle Interaction.

Maki Kamoshita, Michael Schrader.

  Peroxisomes are essential organelles in mammals, which contribute to cellular lipid metabolism and redox homeostasis. They do not function as isolated entities but cooperate and interact with other subcellular organelles, in particular the endoplasmic reticulum, mitochondria, and lipid droplets. Those interactions are often mediated by membrane contact sites. Tether proteins at those sites bring the organelles in close proximity to facilitate metabolite and lipid transfer as well as organelle communication. There is great interest in the investigation of the physiological functions of peroxisome-organelle contacts and how they are regulated. Here, we present an antibody- and fluorescence-based proximity ligation approach used successfully in our laboratory for the detection and quantification of peroxisome-organelle interactions in cultured mammalian cells.

Keywords:  Cell culture; Immunofluorescence; Membrane contact sites; Organelle interaction; Peroxisome; Proximity ligation; Transfection

DOI:  https://doi.org/10.1007/978-1-0716-3048-8_10
Front Neurosci. 2023 ;17 1158204

The contribution of mitochondria-associated endoplasmic reticulum membranes (MAMs) dysfunction in Alzheimer's disease and the potential countermeasure.

Zehui Li, Yu Cao, Hui Pei, Lina Ma, Yang Yang, Hao Li.

  Alzheimer's disease (AD) is the most common neurodegenerative disease. There are many studies targeting extracellular deposits of amyloid β-peptide (Aβ) and intracellular neurofibrillary tangles (NFTs), however, there are no effective treatments to halt the progression. Mitochondria-associated endoplasmic reticulum membranes (MAMs) have long been found to be associated with various pathogenesis hypotheses of AD, such as Aβ deposition, mitochondrial dysfunction, and calcium homeostasis. However, there is a lack of literature summarizing recent advances in the mechanism and treatment studies. Accordingly, this article reviews the latest research involving the roles of MAM structure and tethering proteins in the pathogenesis of AD and summarizes potential strategies targeting MAMs to dissect treatment perspectives for AD.

Keywords:  Alzheimer’s disease; calcium homeostasis; endoplasmic reticulum; lipid metabolism; mitochondria

DOI:  https://doi.org/10.3389/fnins.2023.1158204
Neuroscientist. 2023 Mar 24. 10738584231162810

The Endoplasmic Reticulum and Its Contacts: Emerging Roles in Axon Development, Neurotransmission, and Degeneration.

Marijn Kuijpers, Phuong T Nguyen, Volker Haucke.

  The neuronal endoplasmic reticulum (ER) consists of a dynamic, tubular network that extends all the way from the soma into dendrites, axons, and synapses. This morphology gives rise to an enormous membrane surface area that, through the presence of tethering proteins, lipid transfer proteins, and ion channels, plays critical roles in local calcium regulation, membrane dynamics, and the supply of ions and lipids to other organelles. Here, we summarize recent advances that highlight the various roles of the neuronal ER in axonal growth, repair, and presynaptic function. We review the variety of contact sites between the ER and other axonal organelles and describe their influence on neurodevelopment and neurotransmission.

Keywords:  axon; calcium; endoplasmic reticulum; lipids; membrane contact sites; neurodegeneration; neurodevelopment; neuron; neurotransmission

DOI:  https://doi.org/10.1177/10738584231162810
Methods Mol Biol. 2023 ;2643 105-122

Ultrastructural Analysis and Quantification of Peroxisome-Organelle Contacts.

Christian Hacker, Tina A Schrader, Michael Schrader.

  Transmission electron microscopy (TEM) has long been a vital technology to visualize the interaction of cellular compartments at the highest possible resolution. While this paved the way to describing organelles within the cellular context in detail, TEM has long been underused to generate quantitative data, analyzing those interactions as well as underlying mechanisms leading to their formation and modification. Here we describe a simple stereological method to unbiasedly assess the extent of organelle-organelle membrane contact sites, able to efficiently generate accurate and reproducible quantitative data from cultured mammalian cells prepared for TEM.

Keywords:  Cell culture; Electron microscopy; Membrane contact site; Peroxisomes; Quantitation; Stereology; Transmission electron microscopy

DOI:  https://doi.org/10.1007/978-1-0716-3048-8_8
Methods Mol Biol. 2023 ;2643 345-357

Assessing Peroxisomal Protein Interaction by Immunoprecipitation.

Suzan Kors, Michael Schrader.

  Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.

Keywords:  ACBD5; Co-immunoprecipitation; Membrane contact sites; Organelle interactions; Peroxisomes; Phosphorylation; Protein binding assay; Protein interaction; VAPB

DOI:  https://doi.org/10.1007/978-1-0716-3048-8_24
Ecotoxicol Environ Saf. 2023 Apr 01. pii: S0147-6513(23)00214-2. [Epub ahead of print]254 114710

Zearalenone induces mitochondria-associated endoplasmic reticulum membranes dysfunction in piglet Sertoli cells based on endoplasmic reticulum stress.

Li Ma, Sirao Hai, Chenlong Wang, Chuangjiang Chen, Sajid Ur Rahman, Chang Zhao, Mansoor Ahmed Bazai, Shibin Feng, Xichun Wang.

  Zearalenone (ZEA) is an estrogen-like mycotoxin, which mainly led to reproductive toxicity. The study aimed to investigate the molecular mechanism of ZEA-induced dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAM) in piglet Sertoli cells (SCs) via the endoplasmic reticulum stress (ERS) pathway. In this study, SCs were used as a research object that was exposed to ZEA, and ERS inhibitor 4-Phenylbutyrate acid (4-PBA) was used as a reference. The results showed that ZEA damaged cell viability and increased Ca2+ levels; damaged the structure of MAM; up-regulated the relative mRNA and protein expression of glucose-regulated protein 75 (Grp75) and mitochondrial Rho-GTPase 1 (Miro1), while inositol 1,4,5-trisphosphate receptor (IP3R), voltage-dependent anion channel 1 (VDAC1), mitofusin2 (Mfn2) and phosphofurin acidic cluster protein 2 (PACS2) were down-regulated. After a 3 h 4-PBA-pretreatment, ZEA was added for mixed culture. The results of 4-PBA pretreatment showed that inhibition of ERS reduced the cytotoxicity of ZEA against piglet SCs. Compared with the ZEA group, inhibition of ERS increased cell viability and decreased Ca2+ levels; restored the structural damage of MAM; down-regulated the relative mRNA and protein expression of Grp75 and Miro1; and up-regulated the relative mRNA and protein expression of IP3R, VDAC1, Mfn2, and PACS2. In conclusion, ZEA can induce MAM dysfunction in piglet SCs via the ERS pathway, whereas ER can regulate mitochondria through MAM.

Keywords:  Endoplasmic reticulum stress; Mitochondria-associated endoplasmic reticulum membranes; Piglet sertoli cells; Zearalenone

DOI:  https://doi.org/10.1016/j.ecoenv.2023.114710
Metabolism. 2023 Mar 20. pii: S0026-0495(23)00154-3. [Epub ahead of print] 155551

Syntaxin17 contributes to obesity cardiomyopathy through promoting mitochondrial Ca2+ overload in a Parkin-MCUb-dependent manner.

Haixia Xu, Wenjun Yu, Mingming Sun, Yaguang Bi, Ne N Wu, Yuan Zhou, Qi Yang, Mengjiao Zhang, Junbo Ge, Yingmei Zhang, Jun Ren.

   OBJECTIVE: Uncorrected obesity is accompanied by unfavorable structural and functional changes in the heart, known as obesity cardiomyopathy. Recent evidence has revealed a crucial role for mitochondria-associated endoplasmic reticulum membranes (MAMs) in obesity-induced cardiac complication. Syntaxin 17 (STX17) serves as a scaffolding molecule localized on MAMs although its role in obesity heart complication remains elusive.
METHODS AND MATERIALS: This study examined the role of STX17 in MAMs and mitochondrial Ca2+ homeostasis in HFD-induced obesity cardiomyopathy using tamoxifen-induced cardiac-specific STX17 knockout (STX17cko) and STX17 overexpression mice using intravenously delivered recombinant adeno-associated virus serotype-9 (AAV9-cTNT-STX17).
RESULTS: STX17 levels were significantly elevated in plasma from obese patients and heart tissues of HFD-fed mice. Our data revealed that cardiac STX17 knockout alleviated cardiac remodeling and dysfunction in obese hearts without eliciting any notable effect itself, while STX17 overexpression aggravated cardiac dysfunction in obese mice. STX17 deletion and STX17 overexpression annihilated and aggravated, respectively, HFD-induced oxidative stress (O2- production) and mitochondrial injury in the heart. Furthermore, STX17 transfection facilitated obesity-induced MAMs formation in cardiomyocytes and evoked excess mitochondrial Ca2+ influx, dependent upon interaction with mitochondrial Ca2+ uniporter dominant negative β (MCUb) through Habc domain. Our data also suggested that STX17 promoted ubiquitination and degradation of MCUb through the E3 ligase parkin in the face of palmitate challenging.
CONCLUSION: Taken together, our results identified a novel role for STX17 in facilitating obesity-induced MAMs formation, and subsequently mitochondrial Ca2+ overload, mitochondrial O2- accumulation, lipid peroxidation, resulting in cardiac impairment. Our findings denoted therapeutic promises of targeting STX17 in obesity.

Keywords:  MAMs; MCUb; Mitochondrial Ca(2+) overload; Obesity cardiomyopathy; Syntaxin 17

DOI:  https://doi.org/10.1016/j.metabol.2023.155551
Methods Mol Biol. 2023 ;2643 247-270

Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.

Beatriz S C Silva, Tina A Schrader, Michael Schrader, Ruth E Carmichael.

  Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.

Keywords:  CRIS-PITCh; Endogenous tagging; Genome editing; High-throughput analysis; NanoLuc; Peroxisome; Reporter cell line

DOI:  https://doi.org/10.1007/978-1-0716-3048-8_18
Methods Mol Biol. 2023 ;2643 93-104

Correlative Light- and Electron Microscopy in Peroxisome Research.

Rinse de Boer, Ida J van der Klei.

  Correlative light and electron microscopy (CLEM) combines the advantages of protein localization by fluorescence microscopy with the high resolution of electron microscopy. Here, we describe a protocol that we developed for yeast peroxisome research. First, cells are fixed, using conditions that preserve the properties of fluorescent proteins and avoid the introduction of autofluorescence. Next, cryosections are prepared and imaged by fluorescence microscopy. The same sections are used for electron microscopy. Both images are aligned and merged, allowing to localize fluorescent proteins in electron microscopy images. This method was successfully used for peroxisomal membrane contact site research and allows to precisely localize contact site resident proteins at regions where membranes are closely associated at distances far below the resolution of conventional fluorescence microscopy.

Keywords:  Correlative light and electron microscopy; Electron tomography; Fluorescence microscopy; Peroxisome; Transmission electron microscopy; Yeast

DOI:  https://doi.org/10.1007/978-1-0716-3048-8_7