bims-mecosi 2021-10-17 papers

Issue of 2021–10–17
four papers selected by
Verena Kohler, Stockholm University

Eur J Cell Biol. 2021 Oct 04. pii: S0171-9335(21)00031-5. [Epub ahead of print]100(7-8): 151180

Specific subdomain localization of ER resident proteins and membrane contact sites resolved by electron microscopy.

Behnam Lak, Shiqian Li, Ilya Belevich, Sreesha Sree, Rebeka Butkovic, Elina Ikonen, Eija Jokitalo.

The endoplasmic reticulum (ER) is a large, single-copy, membrane-bound organelle that comprises an elaborate 3D network of diverse structural subdomains, including highly curved tubules, flat sheets, and parts that form contacts with nearly every other organelle. The dynamic and complex organization of the ER poses a major challenge on understanding how its functioning - maintenance of the structure, distribution of its functions and communication with other organelles - is orchestrated. In this study, we resolved a unique localization profile within the ER network for several resident ER proteins representing a broad range of functions associated with the ER using immuno-electron microscopy and calculation of a relative labeling index (RLI). Our results demonstrated the effect of changing cellular environment on protein localization and highlighted the importance of correct protein expression level when analyzing its localization at subdomain resolution. We present new software tools for anonymization of images for blind analysis and for quantitative assessment of membrane contact sites (MCSs) from thin section transmission electron microscopy micrographs. The analysis of ER-mitochondria contacts suggested the presence of at least three different types of MCSs that responded differently to changes in cellular lipid loading status.

Keywords: ER subdomains; ER-mitochondria contact sites; Quantitative morphological analysis; Relative labeling index; SOAT1

DOI: https://doi.org/10.1016/j.ejcb.2021.151180

Autophagy. 2021 Oct 13. 1-3

A new type of membrane contact in the ER-Golgi system regulates autophagosome biogenesis.

Shulin Li, Min Zhang, Liang Ge.

Formation of the double-membrane autophagosome requires membrane reorganization of the endomembrane system to generate membrane precursors. The ER-Golgi trafficking system has been shown to provide membranes for phagophore growth. Nonetheless, how the components of the ER-Golgi system are redirected toward autophagosome biogenesis remains unclear. Here, we identify a new type of membrane contact formed between the ER-Golgi intermediate compartment (ERGIC) and the ER-exit sites (ERES) under macroautophagy/autophagy-induction conditions. The ERGIC-ERES contact is established by the TMED9-PREB/SEC12 interaction and regulates the biogenesis of the ERGIC-COPII vesicles, which we found previously act as a membrane template for LC3 lipidation and autophagosome formation.

Keywords: Autophagosome; COPII; ERES; ERGIC; SEC12; TMED9; autophagy

DOI: https://doi.org/10.1080/15548627.2021.1972406

mBio. 2021 Oct 12. e0218021

The Polar Legionella Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane.

Désirée Böck, Dario Hüsler, Bernhard Steiner, João M Medeiros, Amanda Welin, Katarzyna A Radomska, Wolf-Dietrich Hardt, Martin Pilhofer, Hubert Hilbi.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the "Legionella-containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L. pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane in situ. IMPORTANCE Legionnaires' disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L. pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the "Legionella-containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of Legionella-LCV contact sites.

Keywords: Legionella pneumophila; Legionnaires’ disease; bacterial pathogenesis; cryo-electron tomography; pathogen vacuole; type IV secretion system

DOI: https://doi.org/10.1128/mBio.02180-21

Mol Metab. 2021 Oct 09. pii: S2212-8778(21)00201-5. [Epub ahead of print] 101354

An anaplerotic approach to correct the mitochondrial dysfunction in ataxia-telangiectasia (A-T).

A J Yeo, G N Subramanian, K L Chong, M Gatei, R G Parton, D Coman, M F Lavin.

OBJECTIVE: ATM, the protein defective in the human genetic disorder, ataxia telangiectasia (A-T) plays a central role in the response to DNA double strand breaks (DSBs) and in protecting the cell against oxidative stress. We recently showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress.
METHODS: We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2 deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 hours using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS to determine altered levels of TCA intermediates A-T cells compared to healthy control cells.
RESULTS: We demonstrate here that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate.
CONCLUSIONS: These data together show that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has great potential as a novel therapeutic agent for patients with A-T.

Keywords: ATM; Ataxia-telangiectasia; endoplasmic reticulum; heptanoate (C7); mitochondrial dysfunction; mitochondrial interaction; nutrient deprivation

DOI: https://doi.org/10.1016/j.molmet.2021.101354