Biology subjects

Verraes, A.

Publications and source records attributed to Verraes, A..

4 recordsLinked to original sources

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↗

bioRxiv2025

A genetic screen reveals a key role for Reg1 in 2-deoxyglucose sensing and yeast AMPK inhibition

The yeast Saccharomyces cerevisiae thrives in sugar-rich environments by rapidly consuming glucose and favoring alcoholic fermentation. This metabolic strategy is tightly regulated by glucose signaling pathways, particularly the glucose repression pathway, which prevents the expression of genes required for the utilization of alternative carbon source. Central to this regulatory network is the yeast ortholog of the heterotrimeric 5'AMP-activated protein kinase (AMPK), which adjusts gene expression in response to glucose availability. The activity of the yeast AMPK complex is primarily regulated by the phosphorylation state of its catalytic subunit Snf1, a process orchestrated by a balance between upstream kinases and phosphatases. Among the latter, the Protein Phosphatase 1 (PP1) complex Reg1/Glc7 plays a critical role in repressing Snf1 activity under glucose-rich conditions. Despite its importance, the precise mechanism by which glucose availability leads to Snf1 inhibition remains incompletely understood. Evidence suggests that hexokinase 2 (Hxk2) participates in this pathway, potentially coupling the early steps of glucose metabolism to Snf1 signaling. Notably, the toxic glucose analog 2-deoxyglucose (2DG)- which is phosphorylated by Hxk2 but not further metabolized-mimics glucose in its ability to repress Snf1, implicating glucose or 2DG phosphorylation as a key regulatory signal. In this study, we explore the molecular basis of 2DG resistance in yeast through a large-scale genetic screen. The identified mutations confer resistance either by reducing 2DG phosphorylation (e.g., mutations in HXK2) or by enhancing constitutive Snf1 activity, via gain-of-function alleles in AMPK subunits or loss-of-function mutations in REG1 and GLC7. We describe a novel class of REG1 missense mutations, including reg1-W165G, that maintain normal basal Snf1 activity but fail to mediate 2DG-induced Snf1 inhibition, rendering cells effectively immune to 2DG toxicity. These findings position Reg1 as a central mediator in glucose sensing and further suggest that 2DG toxicity primarily results from aberrant Snf1 inactivation rather than a direct metabolic disturbance.

cell biology↗

bioRxiv2022

The ER tether VAPA is required for proper cell motility and for anchoring ER-plasma membrane contact sites to focal adhesions

Cell motility processes highly depend on the membrane distribution of Phosphoinositides (PInst), giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER membrane-resident contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, and is essential to stabilize and anchor ventral ER-PM contact sites to FA, thus mediating microtubule-dependent FA disassembly. To conclude, our results reveal unprecedented functions for VAPA-mediated membrane contact sites during cell motility and provides a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.

cell biology↗

bioRxiv2018

Multiple signaling pathways converge onto the regulation of HAD-like phosphatases to modulate cellular resistance to the metabolic inhibitor 2-deoxyglucose

Cancer cells display an altered metabolism with an increased glycolysis and glucose uptake. Anti-cancer strategies targeting glycolysis through metabolic inhibitors have been considered. Particularly, the glucose analogue 2-deoxyglucose (2DG) is imported into cells and phosphorylated into 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Recent data suggest that 2DG has additional effects in the cell, and resistance to 2DG has also been observed. Using yeast as a model, we engaged an unbiased, mass-spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms. This revealed that two 2DG-6-phosphate phosphatases, Dog1 and Dog2, are induced upon exposure to 2DG and participate in 2DG detoxication. 2DG induces Dog2 by upregulating several signaling pathways, such as the MAPK (Hog1/p38)-based stress-responsive pathway, the Unfolded Protein Response (UPR) triggered by 2DG-induced ER stress, and the MAPK (Slt2)-based Cell Wall Integrity pathway. Thus, 2DG-induced interference with cellular signaling rewires the expression of these endogenous phosphatases to promote 2DG resistance. Consequently, loss of the UPR or CWI pathways leads to hypersensitivity to 2DG. In contrast, DOG2 is transcriptionally repressed by glucose availability in a Snf1/AMPK-dependent manner, and mutants impaired in this pathway are 2DG-resistant. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression is a common strategy to achieve 2DG resistance. The human Dog2 orthologue, HDHD1, also displays 2DG-6-phosphate phosphatase activity in vitro, and its overexpression confers 2DG resistance in HeLa cells, which has important implications for potential future chemotherapies involving 2DG.

genetics↗