Two distinct functions for PI3-kinases in macropinocytosis

Abstract

SummaryClass-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P 3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P 3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P 3 , with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.

Uses and abuses of macropinocytosis

“…The evidence for this includes the observation of PIP3 patches in the over-sized macropinosomes of axenic strains of Dictyostelium (Parent et al, 1998, and see below), which coincide with patches of active Ras (Sasaki et al, 2007;Hoeller et al, 2013), as well as the patches of active Ras and PIP3 that become prominent in circular ruffles (Araki et al, 2007;Welliver and Swanson, 2012). These patches must be maintained despite the expected rapid diffusion of Ras and PIP3 in the plasma membrane, and most likely depend on autocatalytic Ras activation and PIP3 production, possibly combined with the establishment of a diffusion barrier around macropinosomes (Welliver et al, 2011).…”

“…The injection of activated (oncogenic) Ras protein into fibroblasts causes ruffling and macropinocytosis (Bar-Sagi and Feramisco, 1986; see also below). Genetic ablation of certain Ras proteins inhibits macropinocytosis in Dictyostelium amoebae (Chubb et al, 2000;Hoeller et al, 2013), and loss of the Ras GTPase-activating protein (RasGAP) NF1 increases the frequency of macropinocytosis and size of macropinosomes in these cells (Bloomfield et al, 2015; see also below). Ras activates class-I phosphatidylinositol 3-kinases (PI3Ks) through their Rasbinding domain, and these enzymes produce the membrane lipid PIP3 (Rodriguez-Viciana et al, 1994), which is an ether-linked plasmanyl inositol in Dictyostelium (Clark et al, 2014).…”

“…Ras activates class-I phosphatidylinositol 3-kinases (PI3Ks) through their Rasbinding domain, and these enzymes produce the membrane lipid PIP3 (Rodriguez-Viciana et al, 1994), which is an ether-linked plasmanyl inositol in Dictyostelium (Clark et al, 2014). When PIP3 production is attenuated either with drugs or genetically, macropinocytosis is inhibited to striking extents (Araki et al, 2007(Araki et al, , 1996Buczynski et al, 1997;Hoeller et al, 2013;Zhou et al, 1998). In both mammalian cells and Dictyostelium, PIP3 is lost from macropinocytic vesicles shortly after they close, giving rise first to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P 2 ], which (in mammalian cells at least) is then converted to phosphatidylinositol 3-phosphate as the vesicle progresses through the endocytic pathway (Araki et al, 2007;Dormann et al, 2004;Maekawa et al, 2014;Swanson, 2014;Welliver and Swanson, 2012;Yoshida et al, 2009;Egami et al, 2014).…”

ADiaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

“…Comparable to mammalian cells, the initial steps of the underlying signaling cascades are quite well understood and are initiated by G protein-coupled folate receptor fAR1 activation upstream of heterotrimeric G proteins (12,13). This step is followed by activation of small GTPases of the Ras family, which act as master regulators of diverse downstream signaling pathways, including target of rapamycin complex 2 (14) and class-I PI3-kinases (PI3Ks) (15), albeit Dictyostelium cells use ether-linked plasmanylinositides instead of phosphatidylinositides (16). PI3K1 and PI3K2 produce patches of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), which are linked to actin-dependent cup formation, whereas PI3K4 is required for the conversion of the cups into intracellular vesicles (15,17).…”

Mechanism of Superoxide Mediated Regulation of Particle Uptake and Exocytosis by a GPI-anchored Superoxide Dismutase C in Dictyostelium

“…5 and Fig.6), and hence macropinocytosis is the main fluid uptake mechanism in Dictyostelium cells (Hoeller et al, 2013) hours and measured exocytosis subsequently every twenty minutes for two hours using a spectrofluorimeter, as described in the Methodology.…”