Biology subjects

Sauvanet, C.

Publications and source records attributed to Sauvanet, C..

6 recordsLinked to original sources

bioRxiv2025

ORP5 controls the partitioning of phosphatidic acid between triacylglycerol and cardiolipin synthesis at mitochondria-ER-lipid droplet contact sites

Phosphatidic acid (PA) is a central metabolic intermediate that can fuel triacylglycerol (TAG) synthesis in lipid droplets (LDs) or cardiolipin production in mitochondria, but how cells partition PA between these competing fates has remained a fundamental unresolved question in lipid cell biology. We identify the lipid transfer protein ORP5 as a key regulator of PA partitioning at Mitochondria-Associated endoplasmic reticulum Membranes (MAM) that contact lipid droplets (LD), referred to as MAM-LD junctions. Cell imaging analysis shows that ORP5 stabilizes PA levels at MAM to promote TAG synthesis. On the other hand, loss of ORP5 causes PA accumulation on mitochondrial membranes, leading to excess cardiolipin synthesis and mitochondrial hyperfusion, while impairing triacylglycerol (TAG) synthesis and LD formation. Finally, reconstitution assays using liposomes or giant organelles further demonstrate that ORP5 can transfer PA from mitochondria to the ER via its ORD domain. Together, these findings reveal that ORP5 functions as a PA lipid transfer protein at tripartite MAM-LD contacts, where it balances LD formation with mitochondrial lipid metabolism, protecting mitochondria from cardiolipin overload.

cell biology↗

bioRxiv2022

Characterization of heterogeneity in nanodisc samples using Feret signatures

Nanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/501900v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19ea37aorg.highwire.dtl.DTLVardef@1fed1aeorg.highwire.dtl.DTLVardef@781962org.highwire.dtl.DTLVardef@ca7859_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LINew methodology to characterize nanodiscs based on Feret signatures C_LIO_LIFeret signatures distinguish nanodisc morphologies and compositions C_LIO_LIAnalysis is highly sensitive to sample quality C_LIO_LIMethod selected condition to solve structure of small membrane protein Bcl-xL C_LI

molecular biology↗

bioRxiv2022

ACE2 nanoparticles prevent cell entry of SARS-CoV-2

The continual evolution of SARS-CoV-2 has challenged the efficacy of many COVID19 vaccines and treatment options. One strategy that evades viral escape is using the entry receptor, human Angiotensin-Converting Enzyme 2 (hACE2). Soluble hACE2 receptor domains show potential as decoys but genetic modifications are necessary to provide sufficient efficacy. However, these engineered constructs are potentially susceptible to viral escape. We combined native hACE2 with viral vectors to form nanoparticles presenting hACE2 analogous to human cells. Cell-based viral infection assays and cryogenic in-situ tomography show that hACE2 nanoparticles sequester viruses through aggregation, efficiently blocking entry of SARS-CoV-2 and its variants in model cell systems and human respiratory tract explants using native hACE2. Thus, we show that hACE2 nanoparticles have high potential as pan-variant COVID19 therapeutics.

cell biology↗

bioRxiv2021

ORP5 AND ORP8 ORCHESTRATE LIPID DROPLET BIOGENESIS AND MAINTENANCE AT ER-MITOCHONDRIA CONTACT SITES

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the Endoplasmic Reticulum (ER). The ER-protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at Mitochondria-Associated ER Membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulate seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for the ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at membrane contact sites. HIGHLIGHTSORP5 and ORP8 localize at MAM subdomains where LDs originate. Phosphatidic acid is enriched in MAM subdomains that are the birthplace of LDs. ORP5 and ORP8 knockdown impairs LD biogenesis. ORP5 and ORP8 regulate seipin recruitment to MAM-LD contact sites.

cell biology↗

bioRxiv2020

LOK/SLK kinases and ERM proteins function together to regulate apical morphology and inhibit RhoA signaling

Activated Ezrin-Radixin-Moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologs LOK and SLK. CRISPR/Cas9 was used to knockout all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM activating phosphorylation. The apical domain of cells lacking LOK/SLK or ERMs is strikingly similar and selectively altered, with loss of microvill, and junctional actin replaced by ectopic myosin-II containing apical stress-fiber-like structures. Constitutively active ezrin can reverse the phenotypes of either ERMs or LOK/SLK knockouts, showing that the major function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

cell biology↗

bioRxiv2019

ORP5 REGULATES TRANSPORT OF LIPIDS AND CALCIUM TO MITOCHONDRIA AT ENDOPLASMIC RETICULUM-MITOCHONDRIA MEMBRANE CONTACT SITES

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at sites of close organelle apposition called membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membranes (MAMs) subdomains, physically linked to the MIB/MICOS complexes that bridge the two mitochondrial membranes. We also show that ORP5/8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a novel physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membranes contacts maintained by the MIB/MICOS complexes.

cell biology↗