Biology subjects

Dubreuil, B.

Publications and source records attributed to Dubreuil, B..

7 recordsLinked to original sources

bioRxiv2025

A multiplexed approach for genetic screening of human cells by electron microscopy uncovers a critical effector of mitochondrial cristae shape

Mitochondria form complex and diverse membrane-architectures essential for their multiple functions. Whereas critical proteins sculpting mitochondrial membranes have been identified, the molecular basis for many key features remains enigmatic. Exploration of membrane ultrastructure, in general, is limited by the tradeoff between resolution and throughput: electron microscopy (EM) is essential to resolve their ultrastructure but lacks scalability for systematic functional discovery. To overcome this limitation, we developed a high-resolution screening pipeline for EM of multiplexed human cell pools, hMultiCLEM (human Multiplexed Correlative Light and EM). To showcase the power of hMultiCLEM we performed a genetic screen exploring mitochondrial ultrastructure. hMultiCLEM confirmed proposed cristae modulators and uncovered additional ones illuminating the protein networks driving cristae organization. Validation of candidates highlighted an intermembrane space (IMS) protein linked to Menieres disease, which we named MISHA (Mitochondrial-IMS membrane-SHApe-impacting protein). More broadly, hMultiCLEM transforms the EM field, enabling genetic/chemical screening in basic and medical research.

cell biology↗

bioRxiv2025

A pipeline for screening condition-specific enzymes uncovers a function for the alcohol dehydrogenase Bdh2

Cells possess intricate metabolic networks comprised of hundreds of enzymes. Despite extensive research, many of these enzymes remain uncharacterized. Identifying such enzymes is crucial for advancing our understanding of metabolism. However, multiple enzymes are not expressed in standard conditions, making them challenging to study. To overcome this challenge, we created a pipeline for characterizing the expression of condition-specific enzymes in yeast. We assembled a collection of 110 yeast strains, each containing an uncharacterized putative enzyme fused to a fluorophore under the regulation of their own promotor. By subjecting them to 43 diverse growth or stress environments, we identified the biologically relevant conditions for the expression of 19 proteins. We focused on one such putative alcohol dehydrogenase, Bdh2, and functionally characterized it. More broadly, our discovery pipeline lays the foundation for uncovering new condition-specific enzymes. This has implications in cell biology and biotechnology and should expand our understanding of metabolism.

cell biology↗

bioRxiv2025

Creation and Validation of a Proteome-Wide Yeast Library for Protein Detection and Analysis

A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome- wide collections featuring various fused proteins, the impact of tag size on protein function highlighted the need for using minimally disruptive tags for functional genomic studies. To rise to this challenge, we have created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the Hemagglutinin (HA) epitope. The library leverages the compact size of the HA tag to minimize drawbacks associated with larger tags while enabling efficient functional analysis. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.

molecular biology↗

bioRxiv2024

The phase separation landscape of genome-wide genetic perturbations

Biomolecular organization is central to cell function. While phase separation is a key mechanism orchestrating this organization, we lack a comprehensive view of genes that can globally influence this process in vivo. To identify such genes, we combined functional genomics and synthetic biology. We developed a bioorthogonal system that can identify changes in the intracellular milieu that globally tune phase separation. We measured in vivo phase diagrams of a synthetic system across >25 million cells in 2,888 yeast knockouts, and identified 68 genes whose deletion alters the phase boundaries of the synthetic system, an unexpected result given the systems bioorthogonal design. Genes involved in TORC1 signaling and metabolism, particularly carbohydrate-, amino acid- and nucleotide synthesis were enriched. The mutants that changed phase separation also showed high pleiotropy, suggesting that phase separation interrelates with many aspects of biology. Highlights- A synthetic protein system reveals the genetic and environmental tunability of protein phase separation - Genetic knockouts affecting phase separation are highly pleiotropic - Carbohydrate, amino acid, and nucleotide metabolism contribute to modulating phase separation potential - Protein phase separation is a globally tunable property of the intracellular environment O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/620319v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@4b8403org.highwire.dtl.DTLVardef@1c80503org.highwire.dtl.DTLVardef@c10bf2org.highwire.dtl.DTLVardef@1f76c71_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

A proteome-wide yeast degron collection for the dynamic study of protein function.

Genome-wide collections of yeast strains, known as libraries, revolutionized the way systematic studies are carried out. Specifically, libraries that involve a cellular perturbation, such as the deletion collection, have facilitated key biological discoveries. However, short-term rewiring and long-term accumulation of suppressor mutations often obscure the functional consequences of such perturbations. We present the AID library which supplies "on demand" protein depletion to overcome these limitations. Here, each protein is tagged with a Green Fluorescent Protein (GFP) and an Auxin inducible degron (AID), enabling rapid protein depletion that can be quantified systematically using the GFP element. We characterized the degradation response of all strains and demonstrated its utility by revisiting seminal yeast screens for genes involved in cell cycle progression as well as mitochondrial distribution and morphology. In addition to recapitulating known phenotypes, we also uncovered proteins with previously unrecognized roles in these central processes. Hence, our tool expands our knowledge of cellular biology and physiology by enabling access to phenotypes that are central to cellular physiology and therefore rapidly equilibrated.

cell biology↗

bioRxiv2023

An atlas of protein homo-oligomerization across domains of life

Protein structures are essential to understand cellular processes in molecular detail. While advances in AI revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. Here, we describe a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. We find that 50% of archaeal, 45% of bacterial, and 20% of eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of novel homo-oligomer types. Analyzing these datasets reveals coiled-coil regions as major enablers of quaternary structure evolution in Eukaryotes. Integrating these structures with omics data shows that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations, which we find enriched at interfaces. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes, protein networks, and disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/544317v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1507a12org.highwire.dtl.DTLVardef@7e522aorg.highwire.dtl.DTLVardef@1445410org.highwire.dtl.DTLVardef@eb09f7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2019

Proteomic analysis reveals the recruitment of intrinsically disordered regions to stress granules

Heat-stress triggers the formation of condensates known as stress granules (SGs), which store non-translating mRNA and stalled translation initiation complexes. To gain a better understanding of SGs, we identified yeast proteins that sediment after heat-shock by mass spectrometry. Heat-regulated proteins are biased toward a subset of abundant proteins that are significantly enriched in intrinsically disordered regions (IDRs). SG localization of over 80 heat-regulated proteins was confirmed using microscopy, including 32 proteins that were not known previously to localize to SGs. We find that several IDRs are sufficient to mediate SG recruitment. Moreover, the diffusive exchange of IDRs within SGs, observed via FRAP, can be highly dynamic while other components remain immobile. Lastly, we showed that the IDR of the Ubp3 deubiquitinase is critical for SG formation. This work confirms that IDRs play an important role in cellular compartmentalization upon stress, can be sufficient for SG incorporation, can remain dynamic in vitrified SGs, and play a vital role during heat-stress.\n\nSummaryThe authors provide an in-depth proteomic study of yeast heat stress granule (SG) proteins. They identified intrinsic disordered regions (IDRs) as one of the main features shared by these proteins and demonstrated IDRs can be sufficient for SG recruitment.

cell biology↗