Septins regulate kinase-inhibitor induced micron-scale vacuolation

Several kinase inhibitors including pyridinyl imidazole class p38 MAPK inhibitors and specific PIKFYVE inhibitors have been shown to induce endosomal swelling and micron-scale vacuolation, by inhibiting a PIKFYVE-dependent pathway. We performed a screen to identify small molecule modulators of micron-scale vacuolation and identified septin inhibitor Forchlorfenuron (FCF) as an inhibitor of vacuolation. FCF inhibited vacuolation induced by SB202190, PIKFYVE inhibitors and VE-821. shRNA-mediated depletion of SEPT9 suppressed kinase inhibitor-induced vacuolation, while SEPT7 knockdown did not affect vacuolation. Similar results were obtained when experiments were performed using penfluridol as another modulator of septin cytoskeleton. The kinase-inhibitor induced vesicles, identified as swollen RAB7+ late endosomes colocalized with CD63 and FCF treatment led to the loss of these RAB7-labelled micron-scale vacuoles. FCF not only abrogates vacuole formation but also suppresses resolution of vacuoles upon SB202190 withdrawal. However, septin filaments do not colocalize with the vacuoles. Unlike bafilomycin, which inhibits vacuolation with parallel blockade of autophagic proteolysis, FCF mediated suppression of vacuoles doesn't involve accumulation of autophagy markers. The role of septins in micron scale vacuolation may be linked to their role in endosome maturation and septins may contribute towards the cell-type specificity of micron-scale vacuolation.