Identification of DYRK1b as a novel regulator of small extracellular vesicle release using a high throughput nanoscale flow cytometry screening platform

Extracellular vesicles (EVs) are important mediators of intercellular communication and have various roles in physiological and pathological processes. Discovery of regulators of EV biogenesis and release has led to significant improvements in our understanding of EV biology and has highlighted disease-specific pathways. Large scale discovery studies of EV regulators are limited by conventional methods of EV analysis with limited throughput and sensitivity. To address this, this study presents a high-throughput flow cytometry-based platform for the quantification of EVs released from cells. Here, a system was developed using the MDA-MB-231 cell line stably expressing ZsGreen, which passively loads ZsGreen proteins into EVs, and nanoscale flow cytometry. EV detection and quantitation was optimized and validated for a 96-well format. The high-throughput flow cytometry screening platform quantified the effect of 156 kinase inhibitors on EV number and identified AZ191 - a DYRK1b inhibitor - as a potent EV inhibitor. DYRK1b inhibition and knockdown confirmed a significant reduction in total EV number, with small EVs demonstrating the largest reduction. DYRK1b knockdown altered the intracellular distribution of EV marker CD63, suggesting a role for DYRK1b in EV trafficking. In conclusion, our study establishes a platform for high-throughput analysis of EV dynamics and introduces DYRK1b kinase as a novel EV-regulator.