The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis

Abstract

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain PI(4,5)P2 homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated in ER cisternae juxtaposed to phagocytic cups when expressed in phagocytic mouse fibroblasts. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE), and receptor-mediated phagocytosis, stalling particle capture at cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2/3-edited cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 editing reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir-mediated lipid transfer maintains phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis. Summary statementChanges in membrane phosphoinositides coordinate actin remodeling during phagocytosis, but whether lipid transport proteins contribute to this process is not known. Here, we show that the phosphatidylinositol transfer proteins Nir2 and Nir3 are recruited to phagocytic cups and drive the formation of contractile actin rings during particle engulfment. Using gene editing and re-expression, we show that Nir2 and Nir3 maintain PI(4,5)P2 signaling competence at phagocytic cups and promote the actin-dependent sealing of phagocytic vacuoles. These observations establish that lipid transport proteins maintain the phosphoinositide signals that drive the remodeling of the actin cytoskeleton during phagocytosis.