CD24 regulates extracellular vesicle release via an aSMase/PI3K/mTORC2/ROCK/actin pathway in B lymphocytes
CD24 is a glycophosphatidylinositol-linked protein that regulates B cell development. We previously reported that stimulation of CD24 on donor B cells promotes the transfer of functional receptors to recipient B cells via extracellular vesicles (EVs). However, the mechanisms regulating CD24-mediated formation of bioactive EVs are unknown. Using bioinformatics, we found a connection between CD24, and PI3K/AKT and mTOR. To determine if these pathways regulate EV release, we used flow cytometry to follow the transfer of EVs carrying lipid- associated GFP and surface IgM from donor to recipient B cells. Using chemical and genetic inhibition, we found that a PI3K/mTORC2/ROCK/actin pathway regulates bioactive EV formation via activation of acid sphingomyelinase (aSMase) upstream of PI3K. Using single EV analysis, we found that CD24 regulates the formation of the subset of bioactive EVs that are taken up by recipient cells and not total EVs. Interestingly, we also found that ROCK and aSMase modulate ectosome but not exosome formation, when CD24 is stimulated. Lastly, through live cell imaging, we found that PI3K and ROCK are required for inducing membrane dynamics associated with EV formation. These data suggest that this pathway regulates bioactive EV release that, in turn, could regulate B cell development. Graphical TOC/AbstractO_LIBioinformatics analysis followed by genetic and chemical experiments revealed that a novel pathway regulates EV release downstream of CD24; namely aSMase/PI3K/mTORC2/ROCK/actin. C_LIO_LISingle EV analysis suggests that the EVs are ectosomes and that CD24 regulates packaging of cargo to create bioactive vesicles rather than regulating total EV release. C_LIO_LILive cell imaging shows that CD24 regulates membrane dynamics. C_LI O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/607772v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@262ac2org.highwire.dtl.DTLVardef@1c150b0org.highwire.dtl.DTLVardef@98779dorg.highwire.dtl.DTLVardef@1d51803_HPS_FORMAT_FIGEXP M_FIG C_FIG