Thiagarajan, S. | bioRxiv Science

bioRxiv2026

Glomerular Endothelial Cell-Derived Extracellular Vesicles Cross the Basement Membrane to Regulate Podocyte Function

BackgroundSmall extracellular vesicles (EVs) are nanosized, endosome-derived particles which transfer RNA, proteins, and bioactive molecules to mediate intercellular communication. While EV signaling has been observed in many organ systems, it remains unclear whether glomerular endothelial cell (GEC)-derived small EVs directly interact with podocytes in vivo or how they traverse the glomerular basement membrane (GBM). MethodsGEC-derived small EVs were characterized by nanoparticle tracking analysis, electron microscopy, RAMAN spectroscopy and flow cytometry. Cargo composition was analyzed by proteomics, and microRNA (miR) profiling. Functional and structural features were examined using protease, collagenase, adhesion, and multimodal imaging assays. GEC-derived small EV uptake and downstream transcriptional effects were studied in cultured podocytes, while in vivo trafficking was assessed by injection of labeled small EVs into transgenic zebrafish larvae under baseline conditions, puromycin-induced damage, and cd2ap-knockdown. ResultsGECs released bona fide exosome-like small EVs carrying a highly cell type-specific miR cargo. Small EV transfer to podocytes induced a defined transcriptional response consistent with miR-mediated repression of target genes involved in extracellular matrix organization, cell cycle regulation, and cellular stress responses. Proteomic analyses revealed enrichment of surface proteases and integrin-associated proteins that conferred sustained proteolytic activity and enabled GEC-derived small EV migration through extracellular matrix surrogates. In vivo, circulating small EVs traversed the GBM and localized selectively to podocytes in healthy glomeruli, whereas glomerular injury permitted small EV entry into the tubular compartment. ConclusionThese findings provide first in vivo evidence that GEC-derived small EVs can cross the GBM and impact on podocytes. By identifying integrin- and protease-dependent mechanisms which facilitate vesicle passage, this study redefines the GBM as a dynamic interface of heterocellular, vesicle-mediated communication.

physiology↗

bioRxiv2025

Student-led experimental evolution reveals novel biofilm regulatory networks underlying adaptations to multiple niches

We established a research-education partnership, EvolvingSTEM, that currently provides thousands of secondary school students the opportunity to conduct authentic research experiments centered on microbial evolution each year. Providing high school students access to research experiences not only improves learning and can have positive and long-lasting impacts on their attitudes towards science, but also gives them the opportunity to make impactful scientific contributions. Through EvolvingSTEM, students evolve populations of Pseudomonas fluorescens in a bead model that includes daily cycles of bacterial dispersal, attachment, and biofilm growth and observe heritable changes in colony morphology. Genome sequencing of 70 mutants that they picked identified parallel mutations in genes known to regulate biofilm growth (wsp, yfiBNR, morA, fuzY) and uncovered novel adaptations: loss-of-function mutations in phosphodiesterase PFLU0185 that did not alter colony morphology and mutations affecting periplasmic disulfide bond formation producing small colonies. PFLU0185 mutants rapidly and consistently reached high frequencies and phenotyping revealed roles in cyclic di-GMP regulation, biofilm formation, and motility, prompting us to name this gene bmo (biofilm and motility regulator). Competition experiments and microscopy demonstrated bmo mutants employ generalist strategies and coexist with their ancestor and specialist mutants through niche differentiation. Consequently, phenotypic diversity is maintained, with smooth (ancestral and bmo) colonies consistently outnumbering wrinkly and fuzzy variants. This study advances our understanding of biofilm genetic architecture while demonstrating that student-led research can uncover mechanisms of microbial adaptation relevant to Pseudomonas infection biology. IMPORTANCEBacterial biofilms dominate microbial life, yet their evolutionary genetics remain incompletely understood. Extensive replication of experiments that employ similar, but not identical, biofilm selection models can provide valuable insights into mechanisms of adaptation. We demonstrate that this can be achieved through university-education partnerships that engage secondary school students in authentic research. Student-led experiments revealed that loss-of-function mutations in a conserved phosphodiesterase, PFLU0185/bmo, dominate evolved populations without changing colony morphology. This finding, combined with diverse, less frequent mutants that alter colony morphology informs the process of biofilm niche differentiation. This work also demonstrates the power of distributed research networks for discovering new genetic pathways of adaptation. Students gained authentic research experience, potentially inspiring them to become scientists, while identifying mutants adapted to discrete conditions that maintain diversity within biofilms. This synergy between education and discovery offers a scalable model for addressing complex biological questions while developing scientific literacy in diverse classrooms.

microbiology↗