bims-mecosi 2023-06-18 papers

Issue of 2023–06–18
four papers selected by
Verena Kohler, University of Graz

J Cell Sci. 2023 Jun 12. pii: jcs.260786. [Epub ahead of print]

Tethering proteins of the same contact site affect the localization and mobility of each other.

Lucia Amado, Alexandra P Cogan, Ayelén González Montoro.

Membrane contact sites enable the exchange of metabolites between subcellular compartments and regulate organelle dynamics and positioning. These structures often contain multiple proteins that tether the membranes, establishing the apposition and functionalizing the structure. In this work, we use drug-inducible tethers in vivo to address how different tethers influence each other. We find that the establishment of a region of membrane proximity can recruit tethers, influencing their distribution between different localizations or protein complexes. In addition, restricting the localization of one tether to a subdomain of an organelle causes other tethers to be restricted there. Finally, we show that the mobility of contact site tethers can also be influenced by other tethers of the same interface. Overall, our results show that the presence of other tethers at contact sites is an important determinant of the behavior of tethering proteins. This suggests that contact sites with multiple tethers are controlled by the interplay between specific molecular interactions and the cross-influence of tethers of the same interface.

Keywords: Contact sites; Lipid transfer proteins; Organelles; Tethers

DOI: https://doi.org/10.1242/jcs.260786

J Steroid Biochem Mol Biol. 2023 Jun 13. pii: S0960-0760(23)00104-8. [Epub ahead of print] 106349

Systemwide effects of ER-intracellular membrane contact site disturbance in primary endothelial cells.

Juuso H Taskinen, Hanna Ruhanen, Silke Matysik, Reijo Käkelä, Vesa M Olkkonen.

Membrane contact sites (MCS) make up a crucial route of inter-organelle non-vesicular transport within the cell. Multiple proteins are involved in this process, which includes the ER-resident proteins vesicle associated membrane protein associated protein A and -B (VAPA/B) that form MCS between the ER and other membrane compartments. Currently most functional data on VAP depleted phenotypes have shown alterations in lipid homeostasis, induction of ER stress, dysfunction of UPR and autophagy, as well as neurodegeneration. Literature on concurrent silencing of VAPA/B is still sparse; therefore, we investigated how it affects the macromolecule pools of primary endothelial cells. Our transcriptomics results showed significant upregulation in genes related to inflammation, ER and Golgi dysfunction, ER stress, cell adhesion, as well as Coat Protein Complex-I and -II (COP-I, COP-II) vesicle transport. Genes related to cellular division were downregulated, as well as key genes of lipid and sterol biosynthesis. Lipidomics analyses revealed reductions in cholesteryl esters, very long chain highly unsaturated and saturated lipids, whereas increases in free cholesterol and relatively short chain unsaturated lipids were evident. Furthermore, the knockdown resulted in an inhibition of angiogenesis in vitro. We speculate that ER MCS depletion has led to multifaceted outcomes, which include elevated ER free cholesterol content and ER stress, alterations in lipid metabolism, ER-Golgi function and vesicle transport, which have led to a reduction in angiogenesis. The silencing also induced an inflammatory response, consistent with upregulation of markers of early atherogenesis. To conclude, ER MCS mediated by VAPA/B play a crucial role in maintaining cholesterol traffic and sustain normal endothelial functions.

Keywords: VAPA and VAPB; cholesterol; endothelial cells; inflammation; lipidomics; transcriptomics

DOI: https://doi.org/10.1016/j.jsbmb.2023.106349

Nat Commun. 2023 Jun 16. 14(1): 3586

Ratiometric measurement of MAM Ca2+ dynamics using a modified CalfluxVTN.

Eunbyul Cho, Youngsik Woo, Yeongjun Suh, Bo Kyoung Suh, Soo Jeong Kim, Truong Thi My Nhung, Jin Yeong Yoo, Tran Diem Nghi, Su Been Lee, Dong Jin Mun, Sang Ki Park.

Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca2+ crosstalk. Despite the central importance of MAM Ca2+ dynamics in diverse biological processes, directly and specifically measuring Ca2+ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca2+ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca2+-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca2+ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca2+ indicator, MAM-Calflux estimates steady-state MAM Ca2+ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca2+ and the elucidation of abnormally accumulated MAM Ca2+ from the neurons of Parkinson's disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca2+ communication.

DOI: https://doi.org/10.1038/s41467-023-39343-2

Proc Natl Acad Sci U S A. 2023 Jun 20. 120(25): e2219790120

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells.

Martina Barends, Nicole Koller, Christian Schölz, Verónica Durán, Berislav Bošnjak, Jennifer Becker, Marius Döring, Hanna Blees, Reinhold Förster, Ulrich Kalinke, Robert Tampé.

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Keywords: antigen presentation; antigen processing; membrane organization; membrane proteins; primary cells

DOI: https://doi.org/10.1073/pnas.2219790120