bims-mecosi 2021-12-26 papers

Issue of 2021–12–26
five papers selected by
Verena Kohler, Stockholm University

Front Cell Dev Biol. 2021 ;9 789959

Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction.

Sara Benhammouda, Anjali Vishwakarma, Priya Gatti, Marc Germain.

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.

Keywords: contact sites methodologies; endoplasmic reticulum; mitochondia; organelle; organelle contact sites

DOI: https://doi.org/10.3389/fcell.2021.789959

Membranes (Basel). 2021 Dec 09. pii: 971. [Epub ahead of print]11(12):

Membrane Contact Sites in Yeast: Control Hubs of Sphingolipid Homeostasis.

Philipp Schlarmann, Atsuko Ikeda, Kouichi Funato.

Sphingolipids are the most diverse class of membrane lipids, in terms of their structure and function. Structurally simple sphingolipid precursors, such as ceramides, act as intracellular signaling molecules in various processes, including apoptosis, whereas mature and complex forms of sphingolipids are important structural components of the plasma membrane. Supplying complex sphingolipids to the plasma membrane, according to need, while keeping pro-apoptotic ceramides in check is an intricate task for the cell and requires mechanisms that tightly control sphingolipid synthesis, breakdown, and storage. As each of these processes takes place in different organelles, recent studies, using the budding yeast Saccharomyces cerevisiae, have investigated the role of membrane contact sites as hubs that integrate inter-organellar sphingolipid transport and regulation. In this review, we provide a detailed overview of the findings of these studies and put them into the context of established regulatory mechanisms of sphingolipid homeostasis. We have focused on the role of membrane contact sites in sphingolipid metabolism and ceramide transport, as well as the mechanisms that prevent toxic ceramide accumulation.

Keywords: Saccharomyces cerevisiae; ceramides; lipotoxicity; membrane contact sites; metabolism; non-vesicular transport; sphingolipids; yeast

DOI: https://doi.org/10.3390/membranes11120971

J Cell Biol. 2022 Jan 03. pii: e202112057. [Epub ahead of print]221(1):

Thank ORP9 for FFAT: With endosomal ORP10, it's fission accomplished!

Louise H Wong, Andrea Martello, Emily R Eden.

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol.https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER-endosome contact sites, with the consequent recruitment of endosomal fission factors.

DOI: https://doi.org/10.1083/jcb.202112057

Int J Mol Sci. 2021 Dec 09. pii: 13271. [Epub ahead of print]22(24):

Sequestosome 1 Is Part of the Interaction Network of VAPB.

Christina James, Christof Lenz, Henning Urlaub, Ralph H Kehlenbach.

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8). We performed RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC) to identify proteins that interact with or are in close proximity to P56S-VAPB. The mutation abrogates the interaction of VAPB with many known binding partners. Here, we identify Sequestosome 1 (SQSTM1), a well-known autophagic adapter protein, as a major interaction/proximity partner of P56S-VAPB. Remarkably, not only the mutant protein, but also wild-type VAPB interacts with SQSTM1, as shown by proximity ligation assays and co-immunoprecipiation experiments.

Keywords: SQSTM1; VAPB; amyotrophic lateral sclerosis; p62; sequestosome

DOI: https://doi.org/10.3390/ijms222413271