Proux-Gillardeaux, V. | bioRxiv Science

bioRxiv2025

Lipid transfer by ORP3 is required for the regulation of PI4P and PI(4,5)P2 at the plasma membrane in mitosis

During mitosis, cellular contents including the genetic material and membrane-bound organelles must be faithfully distributed between the two daughter cells. Regulation of PI(4,5)P2 levels at the plasma membrane is essential for mitotic progression, including anchoring of the mitotic spindle, recruitment of the actomyosin cytoskeleton at the cleavage furrow, and abscission. Here, we demonstrate that the ORP3 lipid transfer protein, which transfers PI4P from the plasma membrane to the endoplasmic reticulum (ER) at ER-plasma membrane contacts, plays a crucial role in the regulation of PI4P and PI(4,5)P2 levels at the plasma membrane in mitosis. We show that defects in ORP3 function alter PI4P and PI(4,5)P2 distributions, distribution of the actin cytoskeleton at the plasma membrane, mitotic spindle geometry, chromosome segregation, abscission, and lead to the accumulation of multinucleated cells. The function of ORP3 in mitosis is dependent on its ER-partner VAPA and phosphorylation of the ORP3 VAPA-binding motif strongly recruits ORP3 to the ER, priming it for PI4P transfer from the plasma membrane to the ER. Finally ORP3 is required to prevent PI4P accumulation at the cytoplasmic bridge as a result of PI(4,5)P2 hydrolysis for abscission and successful completion of cell division. Altogether, ORP3 plays a key role in PI4P and PI(4,5)P2 regulation during mitosis. Impairment of ORP3 function results in multiple cell division phenotypes, leading to genetic instability and aneuploidy.

cell biology↗

bioRxiv2024

Direct observation of fluorescent proteins in gels: a rapid, cost-efficient and quantitative alternative to immunoblotting

The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green, orange and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

biochemistry↗