Phosphoinositide-3-kinase-independent contractile activities associated with Fcγ-receptor-mediated phagocytosis and macropinocytosis in macrophages

Abstract

Previous studies have shown that Fcγ receptor (FcR)-mediated phagocytosis and macropinocytosis in macrophages consist of two dissociable activities: a phosphoinositide 3-kinase (PI3K)-independent extension of phagocytic cups and a PI3K-dependent contractile mechanism that closes phagosomes and ruffles into intracellular organelles. Here, we identify an additional contractile activity that persists in the presence of the PI3K inhibitor wortmannin. ML-7, an inhibitor of myosin-light-chain kinase (MLCK), inhibited FcR-mediated phagocytosis, macropinocytosis and cell movements associated with ruffling. Scanning electron microscopy demonstrated a striking difference in morphology between phagocytic cups in the different inhibitors: whereas phagocytic cups of control cells and wortmannin-treated cells conformed closely to particles and appeared to have constricted them, the phagocytic cups in cells treated with ML-7 were more open. Video microscopy of macrophages expressing green-fluorescent-protein (GFP)—actin fusions revealed that bound IgG-opsonized erythrocytes were squeezed during phagosome formation and closure. In ML-7, GFP—actin-rich protrusions extended outward but failed to squeeze particles. Moreover, in contrast to the effects of PI3K inhibitors, ML-7 markedly reduced ruffle movement, and perturbed circular ruffle formation. These PI3K-independent myosin-II-based contractile activities that squeeze phagocytic cups and curve ruffles therefore represent a third component activity of the actin cytoskeleton during phagocytosis and macropinocytosis.

Human papillomavirus entry into NK cells requires CD16 expression and triggers cytotoxic activity and cytokine secretion

“…Moreover, VLP internalization [17]. CD16 has been shown to be involved in macropinocytosis in macrophages [37] and in gd T cells [38]. Moreover, transduction of CD16 into a CD8 1 T-cell clone was sufficient to increase HPV-VLP entry into these cells (Supporting Information Fig.…”

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation

“…Co-localization was partial with FITC-Dextran, complete with LY and negative with clathrin, a classical receptor mediated endocytosis marker. 16 Treatment of human iDCs with inhibitors of fluid-phase endocytosis such as dimethyl-amiloride, cytochalasin D, LY294002 and rottlerin 17,18 before Cy3-RNA pulsing dramatically reduced the signal intensity and number of vesicles internalizing Cy3-RNA (Figure 3b) as well as the luciferase activity (Figure 3c). Nearly complete blocking of RNA uptake was achieved with rottlerin, which among the used compounds has the highest selectivity for macropinocytosis and leads to irreversible blockade of the process.…”

The coordination of signaling during Fc receptor-mediated phagocytosis

“…In a macrophage containing, fluorescently labeled actin, such as rhodamine actin or chimeras of actin plus green or yellow fluorescent protein (actin-GFP, actin-YFP), fluorescent actin concentrates in the phagocytic cup and appears to advance over the opsonized erythrocyte as a circumferential band, which visibly squeezes the erythrocyte [2][3][4]. This squeezing activity reflects the interactions between actin and myosin, as inhibitors of myosin light chain kinase, which facilitates myosin contraction, inhibit squeezing [2].…”

“…Proteins implicated in the regulation of actin polymerization include Cdc42, Rac [27,28], Wiscott-Aldrich syndrome protein (WASP) [29], Arp2/3 [30], WASP family verprolin homologous protein (WAVE) [30], ezrin [31,32], vasodilator-stimulated phosphoprotein [19], coronin [33], phospholipase C (PLC) [34], and P21-activated kinase-1 (PAK1) [35]. Contractile proteins and their regulators include myosins IC, II, V, IXb [31,36], and X [37], RhoA [38], and myosin light-chain kinase [2]. Proteins that regulate membrane fusion or fission include dynamin 2 [39], amphiphysin IIm [40], Arf6 [41,42], Rab5 [43,44], Rab11 [45], and PLA 2 [46].…”

“…Observations of actin cytoskeleton dynamics on forming phagosomes by fluorescence microscopy indicated a robust and intricate series of regulatory interactions governing the formation of the phagosome [2][3][4]36]. Immunofluorescence microscopy of macrophages fixed at various times during synchronized phagocytosis indicates that different classes of myosin are recruited to phagosomes at different times during the process [31].…”