Biology subjects

Bru, S.

Publications and source records attributed to Bru, S..

4 recordsLinked to original sources

bioRxiv2025

Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol

Cells experience strong variations in the consumption and availability of inorganic phosphate (Pi). Since Pi is an essential macronutrient but excess Pi has negative impacts on nucleotide hydrolysis and metabolism, its concentration must be maintained in a suitable range. Conserved storage organelles, acidocalcisomes, provide this buffering function. We used acidocalcisome-like yeast vacuoles to study how such organelles are set up to for this task. Our combined in vitro and in vivo analyses revealed that their ATP-driven polyphosphate polymerase VTC converts cytosolic Pi into inorganic polyphosphates (polyP), which it transfers into the vacuole lumen. Luminal polyphosphatases immediately hydrolyse this polyP to establish a growing reservoir of vacuolar Pi. Product inhibition by this Pi pool silences the polyphosphatases, caps Pi accumulation, and favours vacuolar polyP storage. Upon cytosolic Pi scarcity, the declining inositol pyrophosphate levels activate the vacuolar Pi exporter Pho91 to replenish cytosolic Pi. In this way, acidocalcisome-like vacuoles constitute a feedback-regulated buffering system for cytosolic Pi, which the cells can switch between Pi accumulation, Pi release, and high-capacity phosphate storage through polyP.

cell biology↗

bioRxiv2025

A Screening Approach Unveils an Unknown Mn2+ - dependent Endopolyphosphatase Activity in Yeast

Inorganic polyphosphate (polyP) is a ubiquitous biopolymer composed of multiple orthophosphates connected by energy-rich phosphoanhydride bonds. In organisms, polyP is digested by two types of enzymes: exopolyphosphatases, which shorten the chain from the ends by cleaving off monophosphate units, and endopolyphosphatases, which cut the chain internally. While several continuous methods are available to monitor exopolyphosphatase activity, endopolyphosphatase activity assays are less common and typically involve multiple tedious steps. Here, we introduce FRET-polyP8, a novel probe for real-time detection of endopolyphosphatase activity. The FRET assay enabled rapid, highly sensitive, single-step detection of specific endopolyphosphatase activity both from isolated proteins and cell extracts. The simple read-out additionally enabled enzyme inhibitor screening. Furthermore, a novel Mn2+-dependent endopolyphosphatase activity in bakers yeast was detected in a quadruple mutant, highlighting the ability to screen for metal-dependence of new endopolyphosphatase activity. This approach thus represents a significant addition to existing methodologies, facilitating the discovery and classification of new endopoly-phosphatases and their inhibitors to advance our understanding of polyP metabolism and regulation.

biochemistry↗

bioRxiv2025

Nuclear Speckle Dynamics are Controlled by Polyphosphate Inhibition of CLK Proteins

Nuclear speckles (NS) are membrane-less nuclear organelles that act as critical hubs for pre-mRNA splicing. Defects in splicing are linked to several human diseases, including cancer, Alzheimers disease, and dystrophies. While CLK kinases regulate the mobilization of splicing factors from NS, the molecular mechanisms underlying NS assembly and dissolution remain unclear. Using an adaptation of the Biotinylation by Antibody Recognition (BAR) technique, we identify polyphosphate (polyP) as a novel and essential regulator of NS dynamics. Polyphosphate, a highly conserved polyanion composed of a chain of phosphate molecules, is involved in several functions in mammalian cells. Here, we show that polyP interacts with the NS core component SRRM2, and polyP depletion disrupts NS organization releasing splicing factors into the nucleoplasm. RNA-seq analysis reveals that polyP depletion increases exon inclusion, particularly in long genes with multiple exons, highlighting its role in splicing regulation. Mechanistically, we demonstrate that polyP acts as a physiological inhibitor of CLK3 kinase, preventing the phosphorylation of SR proteins and thereby maintaining NS stability. Our findings not only expand our understanding of NS biology but also provide new insights into the polyP involvement in splicing-related diseases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/633116v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1dc44b6org.highwire.dtl.DTLVardef@2339e4org.highwire.dtl.DTLVardef@ade454org.highwire.dtl.DTLVardef@69482b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Critical evaluation of kinetic schemes for coagulation

Computational models of the coagulation cascade are used for a wide range of applications in bio-medical engineering such as drug and bio-medical device developments. However, a lack of robustness of numerical models has been highlighted when studying clinically relevant scenarios. In order to develop more robust models, numerical simulations need to be confronted with realistic situations relevant to clinical practice. In this work, two well-established numerical representations of the coagulation cascade initiated by the intrinsic and extrinsic systems, respectively, were compared with thrombin generation assays considering realistic pathological conditions. Proper modifications were needed to align the in vitro and in silico data, namely; adapting initial conditions to the thrombin assay system, omitting reactions irrelevant to our case study, and improving the fitting of some reaction rates. The modified models were able to capture the experimental trends of thrombin generation for a range of concentrations of factors XII, XI, and VIII for cases in which the coagulation cascade is triggered through the extrinsic and intrinsic systems. Our work emphasizes that when existing coagulation cascade models are extrapolated to experimental settings for which they were not calibrated, careful adjustments must be made. We show that the two coagulation models used in this work can predict physiological conditions, but when studying pathological conditions, proper modifications are needed to improve the numerical results.

systems biology↗