Biology subjects

Bouffard, J.

Publications and source records attributed to Bouffard, J..

6 recordsLinked to original sources

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↗

bioRxiv2025

Neuromuscular, Cardiovascular, and Cognitive Fatigue in Motor Learning: A Systematic Review

BackgroundFatigue is multifactorial and task-dependent, arising from the interplay between performance and perceived fatigability. Fatigue-related changes in sensorimotor control and neural activity may alter skill acquisition and retention. ObjectiveTo synthesize behavioral and neurophysiological evidence on how cognitive, cardiovascular, and neuromuscular fatigue influence motor-skill acquisition, consolidation/retention, and transfer. MethodsA systematic search was conducted within PubMed, Web of Science, and Embase. Thirty-four studies (39 experiments) met inclusion criteria (fatigue effects on motor learning in non-disabled participants). Data were extracted on fatigue protocol, learning taxonomy, learning stage (acquisition vs. retention/transfer), interval, and neurophysiology. Methodological quality was appraised with a tailored Downs & Black checklist. ResultsDuring acquisition, 64% of experiments reported impairments under fatigue, particularly for visuomotor precision and fine force control; 10% found facilitation (often after cardiovascular exertion or high cognitive load); 15% were neutral; 10% inconclusive. Retention was tested in a fresh state and in a fatigue state in 34 experiments and 6 experiments, respectively. Results during retention tests were relatively variable, depending on fatigue protocols. Neurophysiological measures were scarce (EEG/TMS in only four studies), and very few studies considered the effects of personal factors such as age or sex. ConclusionsFatigue does not uniformly impair motor learning. Neuromuscular fatigue typically hampers acquisition and sometimes retention, while cardiovascular exertion can transiently prime plasticity and enhance consolidation. Evidence for cognitive fatigue is limited and inconsistent. Future work should use longer retention windows, state-matched testing, and integrated neurophysiology to clarify mechanisms and guide training under fatigue.

neuroscience↗

bioRxiv2019

Caenorhabditis elegans PIEZO Channel Coordinates Multiple Reproductive Tissues to Govern Ovulation

The PIEZO proteins are involved in a wide range of developmental and physiological processes. Human PIEZO1 and PIEZO2 are newly identified excitatory mechano-sensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Calcium signaling in the spermatheca is normal during ovulation in pezo-1 mutants, however, pezo-1 interacts genetically with known regulators of calcium signaling. Lastly, loss of PEZO-1 caused defective sperm navigation after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. These findings suggest that PEZO-1 may act in different reproductive tissues to promote proper ovulation and fertilization in C. elegans.

developmental biology↗

bioRxiv2019

Intercellular communication controls agonist-induced calcium oscillations independently of gap junctions in smooth muscle cells.

We report the existence of a unique mode of communication among human smooth muscle cells (SMCs) where they use force to frequency modulate long-range calcium waves. An important consequence of this mechanical signaling is that changes in stiffness of the underlying extracellular matrix can interfere with the frequency modulation of Ca2+ waves causing healthy SMCs to falsely perceive a much higher agonist dose than they actually received. This distorted sensing of contractile agonist dose on stiffer matrices is absent in isolated SMCs, even though the isolated cells can sense matrix rigidity. We show that intercellular communication that enables this collective Ca2+ response does not involve transport across gap junctions or extracellular diffusion of signaling molecules. The aberrant communication between cells that distorts the individual cell's perception of contractile stimulus can explain the sudden, exaggerated narrowing of the lumen when exposed to low dose of inhaled agonists in diseases like asthma.

biophysics↗

bioRxiv2018

The RhoGAP SPV-1 regulates calcium signaling to control the contractility of the C. elegans spermatheca during embryo transits.

Contractility of the non-muscle and smooth muscle cells that comprise biological tubing is regulated by the Rho-ROCK and calcium signaling pathways. Although many molecular details about these signaling pathways are known, less is known about how they are coordinated spatiotemporally in biological tubes. The spermatheca of the C. elegans reproductive system enables study of the signaling pathways regulating actomyosin contractility in live adult animals. The RhoGAP SPV-1 was previously identified as a negative regulator of RHO-1/Rho and spermathecal contractility. Here, we uncover a role for SPV-1 as a key regulator of calcium signaling. spv-1 mutants expressing the calcium indicator GCaMP in the spermatheca exhibit premature calcium release, elevated calcium levels, and disrupted spatial regulation of calcium signaling during spermathecal contraction. Although RHO-1 is required for spermathecal contractility, RHO-1 does not play a significant role in regulating calcium. In contrast, activation of CDC-42 recapitulates many aspects of spv-1 mutant calcium signaling. Depletion of cdc-42 by RNAi does not suppress the premature or elevated calcium signal seen in spv-1 mutants, suggesting other targets remain to be identified. Our results suggest SPV-1 works through both the Rho-ROCK and calcium signaling pathways to coordinate cellular contractility.\n\nHighlight SummaryThrough in vivo imaging of the calcium sensor GCaMP, we show that the RhoGAP SPV-1 is a key regulator of calcium signaling in the C. elegans spermatheca. Our data suggests SPV-1 acts at least partially through the small GTPase CDC-42 to modulate calcium signaling, while also acting on RHO-1 to modulate Rho-ROCK signaling. This places SPV-1 as a central regulator of cellular contractility.

cell biology↗

bioRxiv2018

Ancestral Roles of the Fam20C Family of Secreted Protein Kinases Revealed by Functional Analysis in C. elegans

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the C. elegans Fam20C ortholog, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains inter-cellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in this context. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.

cell biology↗