bims-mecosi 2023-09-24 papers

Issue of 2023‒09‒24
four papers selected by
Verena Kohler

Nat Commun. 2023 Sep 21. 14(1): 5867

Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites.

Tomoki Naito, Haoning Yang, Dylan Hong Zheng Koh, Divyanshu Mahajan, Lei Lu, Yasunori Saheki.

Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.

DOI: https://doi.org/10.1038/s41467-023-41213-w

Sci Immunol. 2023 Sep 29. 8(87): eabq2424

Mitochondria-ER contact mediated by MFN2-SERCA2 interaction supports CD8+ T cell metabolic fitness and function in tumors.

Metabolic fitness of T cells is essential for their vitality, which is largely dependent on the behavior of the mitochondria. The nature of mitochondrial behavior in tumor-infiltrating T cells remains poorly understood. In this study, we show that mitofusin-2 (MFN2) expression is positively correlated with the prognosis of multiple cancers. Genetic ablation of Mfn2 in CD8+ T cells dampens mitochondrial metabolism and function and promotes tumor progression. In tumor-infiltrating CD8+ T cells, MFN2 enhances mitochondria-endoplasmic reticulum (ER) contact by interacting with ER-embedded Ca2+-ATPase SERCA2, facilitating the mitochondrial Ca2+ influx required for efficient mitochondrial metabolism. MFN2 stimulates the ER Ca2+ retrieval activity of SERCA2, thereby preventing excessive mitochondrial Ca2+ accumulation and apoptosis. Elevating mitochondria-ER contact by increasing MFN2 in CD8+ T cells improves the efficacy of cancer immunotherapy. Thus, we reveal a tethering-and-buffering mechanism of organelle cross-talk that regulates the metabolic fitness of tumor-infiltrating CD8+ T cells and highlights the therapeutic potential of enhancing MFN2 expression to optimize T cell function.

DOI: https://doi.org/10.1126/sciimmunol.abq2424

STAR Protoc. 2023 Sep 15. pii: S2666-1667(23)00525-7. [Epub ahead of print]4(4): 102558

Using DeepContact with Amira graphical user interface.

Liqing Liu, Hongjun Wu, Shuxin Yang, Ke Yi, Junjie Hu, Li Xiao, Tao Xu.

DeepContact is a deep learning software for high-throughput quantification of membrane contact site (MCS) in 2D electron microscopy images. This protocol will guide users through incorporating available DeepContact models in Amira's artificial intelligence module, thereby allowing invoking of DeepContact functions in organelle segmentation and quantifying of MCS with a user-friendly graphical user interface of Amira software. For complete details on the use and execution of this protocol, please refer to Liu et al. (2022).1.

Keywords: Cell Biology; Computer Sciences; Microscopy

DOI: https://doi.org/10.1016/j.xpro.2023.102558

Bio Protoc. 2023 Sep 05. 13(17): e4803

Endoplasmic Reticulum Isolation: An Optimized Approach into Cells and Mouse Liver Fractionation.

Marc Leiro, Raúl Ventura, Nil Rojo-Querol, María Isabel Hernández-Alvarez.

The subfractionation of the endoplasmic reticulum (ER) is a widely used technique in cell biology. However, current protocols present limitations such as low yield, the use of large number of dishes, and contamination with other organelles. Here, we describe an improved method for ER subfractionation that solves other reported methods' main limitations of being time consuming and requiring less starting material. Our protocol involves a combination of different centrifugations and special buffer incubations as well as a fine-tuned method for homogenization followed by western blotting to confirm the purity of the fractions. This protocol contains a method to extract clean ER samples from cells using only five (150 mm) dishes instead of over 50 plates needed in other protocols. In addition, in this article we not only propose a new cell fractionation approach but also an optimized method to isolate pure ER fractions from one mouse liver instead of three, which are commonly used in other protocols. The protocols described here are optimized for time efficiency and designed for seamless execution in any laboratory, eliminating the need for special/patented reagents. Key features • Subcellular fractionation from cells and mouse liver. • Uses only five dishes (150 mm) or one mouse liver to extract highly enriched endoplasmic reticulum without mitochondrial-associated membrane contamination. • These protocols require the use of ultracentrifuges, dounce homogenizers, and/or Teflon Potter Elvehjem. As a result, highly enriched/clean samples are obtained. Graphical overview.

Keywords: Cells; Endoplasmic reticulum (ER); Mitochondrial-associated membranes (MAMs); Mouse liver; Organelle isolation; Subcellular fractionation; Time efficient

DOI: https://doi.org/10.21769/BioProtoc.4803