Biology subjects

Cardinal, A.

Publications and source records attributed to Cardinal, A..

3 recordsLinked to original sources

bioRxiv2025

STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

Membrane contact sites (MCS) are dynamic regions where the membranes of two organelles come into close apposition. MCSs play many roles in cellular homeostasis by facilitating inter-organelle lipid and ion exchange as well as organelle positioning. The late endosome/lysosome (LE/Lys) cholesterol transfer protein STARD3 (StAR-related lipid transfer (START) domain containing protein 3) forms reversible contacts between the LE/Lys and the endoplasmic reticulum (ER). This tether protein contains a Phospho-FFAT motif (two phenylalanines (FF) in an acidic tract (AT)) whose interaction with ER-resident VAP proteins is phosphorylation-dependent. In this study, we identify Glycogen Synthase Kinase 3 (GSK3 and GSK3{beta}) as the kinases responsible for phosphorylating serine 209 within the Phospho-FFAT motif of STARD3. This phosphorylation event is both necessary and sufficient to activate STARD3s tethering activity, thereby promoting ER-LE/Lys contacts. Furthermore, we show that the ER-LE/Lys tethering activity made by STARD3 regulates endosome positioning, revealing an additional function for STARD3 on endosome biology. Our findings establish a direct and critical role for GSK3 in regulating MCS dynamics via STARD3 phosphorylation. This novel insight into GSK3-mediated phosphorylation expands our understanding of the molecular mechanisms governing inter-organelle communication.

cell biology↗

bioRxiv2025

Ist2 promotes lipid transfer by Osh6 via its membrane tethering and lipid scramblase activities

Lipid transfer proteins (LTPs) are required for the uneven distribution of lipids between cellular membranes, which is essential for many cell functions. In yeast, Osh6 is an LTP that exchanges phosphatidylserine (PS) with phosphatidylinositol 4-phosphate (PI(4)P) between the endoplasmic reticulum (ER) and the plasma membrane (PM), promoting the enrichment of PS in the PM. Here, we address why, to function optimally, Osh6 must bind to Ist2, an ER-resident lipid scramblase able to connect the ER to the PM via an intrinsically disordered region (IDR). We determined in vitro that Osh6 binds to the Ist2 IDR with micromolar affinity, whether empty or bound to its lipid ligands. Moreover, we found that Osh6 efficiently transfers PS at ER-PM contact sites if the Ist2 IDR has a minimal length and its binding site in the IDR is sufficiently removed from the ER surface. Next, we reconstituted the Osh6:Ist2 complex within artificial ER-PM contact sites and demonstrated that the association of Osh6 with Ist2 allows for a fast and directed PS flux between the connected membranes. We identified the Ist2 binding site on the Osh6 surface by validating structural models using our functional assays. Finally, we found that the Osh6-mediated PS transfer can be coupled to the PS scramblase activity of Ist2. These data unveil new functional partnerships between an LTP and a membrane tethering/scramblase protein and point to the general advantage of localizing these processes to membrane contact sites to ensure their efficiency.

biochemistry↗

bioRxiv2024

Assessment of salivary microRNA by RT-qPCR: Challenges in data interpretation for clinical diagnosis

Salivary microRNAs (miRNAs) have been recently revealed as the next generation of non-invasive biomarkers for the diagnostics of diverse diseases. However, their short and highly homologous sequences make their quantification by RT-qPCR technique highly heterogeneous and study dependent, thus limiting their implementation for clinical applications. In this study, we evaluated the use of a commercial RT-qPCR kit for quantification of salivary miRNAs for clinical diagnostics. MethodsSaliva was sampled from ten healthy volunteers for a time course analysis. A panel of six miRNA targets (with different sequence homologies) were analysed by one of the most commonly used commercially available RT-qPCR kit. Sensitivity and specificity of the tested miRNA assays were corroborated using synthetic miRNAs. The reliability of all tested assays to differentiate miRNA expression profiles were analysed, to statistically discriminate background noise from intrinsic individual signals. ResultsSignificant variabilities in expression profiles of six miRNAs from ten healthy participants were revealed, yet the poor specificity of the assays offered insufficient performance to associate these differences to biological context. Indeed, as the limit of quantification (LOQ) concentrations are from 2-4 logs higher than that of the limit of detection (LOD), the majority of the analysis for salivary miRNAs felt outside the quantification region. Most importantly, a remarkable number of crosstalk reactions exhibiting considerable OFF target signal intensities was detected, indicating their poor specificity and limited reliability. However, the spike-in of synthetic miRNA increased the capacity to discriminate endogenous salivary miRNA at the LOQ concentrations from those that were significantly lower. ConclusionsOur results demonstrate that comparative analyses for salivary miRNA expression profiles by this commercial RT-qPCR kit are most likely associated to technical limitations rather than to biological differences. In particular, assessment of fundamental parameters including LOD, LOQ and crosstalk of each assay is strictly necessary to interpret observed variations. The standardization of rigorous sample handling and experimental design according to technical parameters of each assay plays a crucial role in reducing data inconsistencies across studies. However, further technological breakthroughs are still required to overcome discrepancies in order to accelerate the translation of salivary miRNAs for clinical applications.

molecular biology↗