CRISPR activation screen uncovers MARCKSL1 as a gauge for extracellular vesicle secretion.
Extracellular vesicles (EVs) are central mediators of intercellular communication that originate from diverse membrane reservoirs. EV biogenesis can occur via multiple pathways, including outward budding of the plasma membrane (PM) and fusion of mature endosomes with the PM. The present study investigates how cells balance these competing routes using a genome-wide CRISPR activation screen for factors that alter surface expression of CD63-an EV-associated tetraspanin that shuttles between the PM and endosomes. This unbiased strategy identifies the membrane adaptor MARCKSL1 known to be upregulated across diverse tumor types. Overexpression of MARCKSL1 elevates CD63 abundance at the PM and boosts EV secretion. Proximity-based proteomics implicates the cytoskeleton-PM linker Radixin and the SNARE-associated protein STXBP3 as MARCKSL1 binding partners that respectively promote either PM-derived or endosome-derived EV secretion. Collectively, our findings reveal new insights into PM remodeling and position MARCKSL1 as a gauge between distinct EV biogenesis platforms.