Biology subjects

Brett, C. L.

Publications and source records attributed to Brett, C. L..

4 recordsLinked to original sources

bioRxiv2026

Human ORFeome expression in S. cerevisiae to better understand extracellular vesicle biology

Extracellular vesicles (EVs) mediate intercellular communication by all organisms studied, from bacteria to yeast to man. Yet evolutionarily conserved mechanisms governing aspects of fundamental EV biology remain enigmatic. To address this, we sought to establish Saccharomyces cerevisiae (bakers yeast) as a model by identifying ectopically expressed human proteins sorted into yeast EVs. Using an optimized pooled cloning method, we inserted >13,000 human open reading frames (ORFs) upstream of an GFP tag within yeast expression plasmids. After transformation into S. cerevisiae, we confirmed expression of 3,288 GFP-tagged human proteins with diverse cellular expression levels and subcellular localizations. Heat stress triggered release of intact, lipid-bound, EGFP-positive small EVs from all transformant pools. Proteomic analysis identified 292 human proteins within EV samples, including canonical human EV biomarkers. Over 70% had yeast orthologs also found in yeast EVs suggesting conserved sorting mechanisms. Protein-protein interaction network analysis linked these EV cargoes to ESCRT-associated pathways. Finally, validation of seven candidates showed that DEF3A, ANXA2 and CLIC1 were enriched in yeast EVs. This study establishes an omics-compatible synthetic biology framework to humanize yeast EVs, begins to uncover conserved cargo sorting mechanisms, and supports future engineering of designer EVs.

synthetic biology↗

bioRxiv2026

Engineering S. cerevisiae extracellular vesicles using synthetic biology

Extracellular vesicles (EVs) hold great promise as therapeutic delivery vehicles, leveraging their natural role as mediators of intercellular communication in all organisms studied. However, many barriers must be overcome to realize their full potential. Saccharomyces cerevisiae is an attractive chassis organism to explore solutions: It is used for drug biomanufacturing, it is amenable to complex genetic engineering, and their EVs can drive responses in human cells. To further develop this prospect, we sought to genetically modify S. cerevisiae EVs by devising a research framework amenable to iterative design, build, test, learn cycles - a core principle of synthetic biology. Using this approach, we focused on identifying new scaffolds - proteins that load cargoes into EVs - from a small pool of candidates. We first optimized a modular cloning strategy, called "EVclo", for plasmid and genome-integrated candidate gene expression. Candidate genes were fused to EGFP, and after confirming expression in cells, we showed that scaffold-EFGP proteins colocalized with mRuby2-tagged Nhx1, a biomarker of multivesicular bodies, presumed sites of EV biogenesis. We triggered release of EVs by heat stress, isolated these EVs by ultrafiltration and size exclusion chromatography, and confirmed the presence of exosome-sized EVs in all samples. We find that candidate scaffold proteins did not affect EV size, morphology or titers. Further analysis of these samples indicated that some EGFP-tagged scaffolds are present in EVs: Bro1, a yeast ortholog of ALIX, was most abundant and ExoSignal showed highest enrichment of the human candidates. In all, we conclude that Bro1 is a good scaffold for future engineering strategies, and that human proteins can be sorted into yeast EVs suggesting conservation of the sorting machinery and demonstrating that yeast EVs can be humanized. This synthetic biology-based, proof-of-concept study establishes S. cerevisiae as a platform to engineer and bioproduce designer EVs for many applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/710173v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@15d407corg.highwire.dtl.DTLVardef@134c916org.highwire.dtl.DTLVardef@7de2beorg.highwire.dtl.DTLVardef@b15348_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS AND TOC BLURBO_LIsynthetic biology-based system was optimized to engineer EVs in S. cerevisiae C_LIO_LIEV scaffolds can be sorted to yeast EVs C_LIO_LIis an efficient scaffold to sort proteins into yeast EVs C_LIO_LIS. cerevisiae can be used to engineer designer EVs for drug delivery C_LI Extracellular vesicles (EVs) are a promising new modality for drug delivery. However, designer EVs must be engineered to broaden applications and improve efficacy. Here, Bouffard et al. optimize methods rooted in synthetic biology to genetically engineer EVs in S. cerevisiae, a yeast commonly used to manufacture biological drugs. They find that ectopically expressed human EV scaffolds (CD63, ExoSignal, PDGFR) can be sorted to yeast EVs, but Bro1 - the yeast ortholog of ALIX - was most efficient at sorting GFP into EVs. This proof-of-concept study demonstrates a single DBTL (design-build-test-learn) cycle that can be used to develop designer EVs for therapeutic applications.

synthetic biology↗

bioRxiv2020

Sphingolipids with Very Long-chain Fatty Acids Regulate Vacuole Fusion During Tethering and Docking

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote robust homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds two carbons to 24-carbon (C24) acyl chains to make C26 VLCFAs that are incorporated into sphingolipids. Vacuoles isolated from elo3{Delta} cells had increased fluidity relative to the wild-type and were attenuated for fusion. Upon further testing we found that vesicle the tethering stage was affected as elo3{Delta} vacuole clusters contained fewer vesicles versus the WT. Vacuole tethering requires the interactions of late endosomal Rab GTPase Ypt7 and the HOPS tethering complex. Pulldown assays using GST-Ypt7 showed that HOPS from elo3{Delta} vacuole extracts failed to bind Ypt7 while HOPS from WT extracts interacted with GST-Ypt7. Furthermore GFP-Ypt7 failed to localize at vertex microdomains of elo3{Delta} vacuoles relative to the WT, whereas HOPS and regulatory lipids did accumulate at vertices. Finally, we found that elo3{Delta} vacuoles had reduced V-ATPase. Together these data show that C26-VLCFA containing sphingolipids are important for Ytp7 function and vacuole homeostasis.

cell biology↗

bioRxiv2018

Acetate and hypertonic stress stimulate organelle membrane fission using distinct phosphatidylinositol signals

Organelle morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying the yeast vacuole as a model, the conserved molecular mechanisms responsible for organelle fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to the cell cycle, osmoregulation or metabolism to change morphology remain elusive. Thus, herein we describe a new fluorometric assay to measure vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a key intermediary metabolite, triggers vacuole fission in vitro and show that it also blocks homotypic vacuole fusion. The basis of this effect is distinct from hypertonic stress, a known trigger of fission and inhibitor of fusion that inactivates the Rab-GTPase Ypt7: Treatment with the phosphatidylinositol-kinase inhibitor wortmannin or the catalytic domain of the Rab-GAP (GTPase Activating Protein) Gyp1 reveal that fission can be triggered by Ypt7 inactivation alone in absence of hypertonic stress, placing it upstream of PI-3,5-P2 synthesis and osmosis required for membrane scission. Whereas acetate seems to block PI-4-kinase to possibly increase the pool of PI on vacuole membranes needed to synthesize sufficient PI-3,5-P2 for fission. Thus, we speculate that both PI-4-P and PI-3-P arms of PI-P signaling drive changes in membrane fission and fusion responsible altering vacuole morphology in response to cellular metabolism or osmoregulation.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC=\"FIGDIR/small/398685_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@896c6dorg.highwire.dtl.DTLVardef@2d8100org.highwire.dtl.DTLVardef@1702e45org.highwire.dtl.DTLVardef@1eddbba_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗