bims-mecosi 2024-03-17 papers

Issue of 2024–03–17
seven papers selected by
Verena Kohler, Umeå University

Cell Death Dis. 2024 Mar 11. 15(3): 203

Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.

Emma L Wilson, Yizhou Yu, Nuno S Leal, James A Woodward, Nikolaos Patikas, Jordan L Morris, Sarah F Field, William Plumbly, Vincent Paupe, Suvagata R Chowdhury, Robin Antrobus, Georgina E Lindop, Yusuf M Adia, Samantha H Y Loh, Julien Prudent, L Miguel Martins, Emmanouil Metzakopian.

Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca2+ release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.

DOI: https://doi.org/10.1038/s41419-024-06568-y

J Transl Med. 2024 Mar 10. 22(1): 261

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

Yi Li, Hua-Bin Wang, Jin-Long Cao, Wen-Jun Zhang, Hai-Long Wang, Chang-Hong Xu, Kun-Peng Li, Yi Liu, Ji-Rong Wang, Hua-Lan Ha, Sheng-Jun Fu, Li Yang.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.
METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.
RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.
CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Keywords: Ischemic reperfusion injury; Kidney; Mass spectrometry; Mitochondria associated membranes; Proteomics analysis

DOI: https://doi.org/10.1186/s12967-024-05021-0

Sci Adv. 2024 Mar 15. 10(11): eadj1512

Exocytic plasma membrane flows remodel endoplasmic reticulum-plasma membrane tethering for septin collar assembly.

Shinju Sugiyama, Keiko Kono.

Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons.

DOI: https://doi.org/10.1126/sciadv.adj1512

Autophagy. 2024 Mar 13.

Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic reticulum in Saccharomyces cerevisiae.

Yang Shi, Kuninori Suzuki.

Macroautophagy/autophagy is the process by which cells degrade their cytoplasmic proteins or organelles in vacuoles to maintain cellular homeostasis under severe environmental conditions. In the yeast Saccharomyces cerevisiae, autophagy-related (Atg) proteins essential for autophagosome formation accumulate near the vacuole to form the dot-shaped phagophore assembly site/pre-autophagosomal structure (PAS). The PAS then generates the phagophore/isolation membrane (PG), which expands to become a closed double-membrane autophagosome. Hereinafter, we refer to the PAS, PG, and autophagosome as autophagy-related structures (ARSs). During autophagosome formation, Atg2 is responsible for tethering the ARS to the endoplasmic reticulum (ER) via ER exit sites (ERESs), and for transferring phospholipids from the ER to ARSs. Therefore, ARS and the ER are spatially close in the presence of Atg2 but are separated in its absence. Because the contact of an ARS with the ER must be established at the earliest stage of autophagosome formation, it is important to know whether the ARS is tethered to the ER. In this study, we developed a rapid and objective method to estimate tethering of the ARS to the ER by measuring the distance between the ARS and ERES under fluorescence microscopy, and found that tethering of the ARS to the ER was lost without Atg1. This method might be useful to predict the tethering activity of Atg2.

Keywords: Autophagy-related 2 (Atg2); Endoplasmic reticulum exit sites (ERES); Fluorescence microscopy; Morphological analysis; Phagophore; Yeast

DOI: https://doi.org/10.1080/15548627.2024.2330033

Life Sci Alliance. 2024 Jun;pii: e202302329. [Epub ahead of print]7(6):

Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

Stephanie M Rockfield, Meghan E Turnis, Ricardo Rodriguez-Enriquez, Madhavi Bathina, Seng Kah Ng, Nathan Kurtz, Nathalie Becerra Mora, Stephane Pelletier, Camenzind G Robinson, Peter Vogel, Joseph T Opferman.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

DOI: https://doi.org/10.26508/lsa.202302329

Planta. 2024 Mar 11. 259(4): 89

Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

Marita Anggarani, Yu-Ying Lin, Shao-An Fang, Hshin-Ping Wu, Chi-Chih Wu, Wann-Neng Jane, Thomas James Roscoe, Frederic Domergue, Yue-Ie Caroline Hsing.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

Keywords: Cork cell; ER-PM contact sites; Epicuticular wax; Epidermal cell; Filament wax; Taiwan oil millet (Eccoilopus formosanus); Wax composition

DOI: https://doi.org/10.1007/s00425-024-04352-y

Ecotoxicol Environ Saf. 2024 Mar 12. pii: S0147-6513(24)00278-1. [Epub ahead of print]274 116202

Polystyrene microplastics impair the functions of cultured mouse Leydig (TM3) and Sertoli (TM4) cells by inducing mitochondrial-endoplasmic reticulum damage.

Giulia Grillo, Sara Falvo, Debora Latino, Gabriella Chieffi Baccari, Massimo Venditti, Maria Maddalena Di Fiore, Sergio Minucci, Alessandra Santillo.

Many laboratory studies demonstrated that the exposure to microplastics causes testosterone deficiency and spermatogenic impairment in mammals; however, the mechanism underlying this process remains still unclear. In this study, we investigated the effects of polystyrene microplastics (PS-MP) on the proliferation and functionality of cultured Leydig (TM3) and Sertoli (TM4) cells, focusing on the mitochondrial compartment and its association with the endoplasmic reticulum (ER). The in vitro exposure to PS-MP caused a substantial reduction in cellular viability in TM3 and TM4 cells. In TM3 cells PS-MP inhibited the protein levels of StAR and of steroidogenic enzymes 3β-HSD and 17β-HSD, and in TM4 cells PS-MP inhibited the protein levels of the androgen receptors other than the activity of lactate dehydrogenase (LDH). PS-MP inhibited the functions of TM3 and TM4, as evidenced by the decrease of the phosphorylation of ERK1/2 and Akt in both cell lines. The oxidative stress caused by PS-MP decreased antioxidant defense in TM3 and TM4 cells, promoting autophagic and apoptotic processes. Furthermore, we found mitochondrial dysfunction and activation of ER stress. It is known that mitochondria are closely associated with ER to form the Mitochondrial-Associated Endoplasmic Reticulum Membranes (MAM), the site of calcium ions transfer as well as of lipid biosynthesis-involved enzymes and cholesterol transport from ER to the mitochondria. For the first time, we studied this aspect in PS-MP-treated TM3 and TM4 cells and MAMs dysregulation was observed. This study is the first to elucidate the intracellular mechanism underlying the effects of PS-MPs in somatic testicular cells, corroborating that PS-MP might be one of the causes of an increase in male infertility through the impairment of steroidogenesis in Leydig cells and of the nurse function of Sertoli cells. Thus, our findings contributed with new information to the mechanism underlying the effects of PS-MP on the male reproductive system.

Keywords: Apoptosis; Endoplasmic reticulum stress; Mitochondrial dysfunction; Polystyrene microplastics; Reproduction; Steroidogenesis

DOI: https://doi.org/10.1016/j.ecoenv.2024.116202