Function of small GTPases inDictyosteliummacropinocytosis

Abstract

Macropinocytosis—the large-scale, non-specific uptake of fluid by cells—is used by Dictyostelium discoideum amoebae to obtain nutrients. These cells form circular ruffles around regions of membrane defined by a patch of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the activated forms of the small G-proteins Ras and Rac. When this ruffle closes, a vesicle of the medium is delivered to the cell interior for further processing. It is accepted that PIP3 is required for efficient macropinocytosis. Here, we assess the roles of Ras and Rac in Dictyostelium macropinocytosis. Gain-of-function experiments show that macropinocytosis is stimulated by persistent Ras activation and genetic analysis suggests that RasG and RasS are the key Ras proteins involved. Among the activating guanine exchange factors (GEFs), GefF is implicated in macropinocytosis by an insertional mutant. The individual roles of Rho family proteins are little understood but activation of at least some may be independent of PIP3.This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

The past, present and future of Dictyostelium as a model system

“…Most recent reviews or relevant papers Cytoskeleton, motility and chemotaxis Gräf et al 2015;Rivero and Xiong, 2016;Stuelten et al 2018;Liu et al 2018;van Haastert et al 2018;Edwards et al 2018;Kriebel et al 2018;Nichols et al 2019;Miyanaga et al, 2018 Phagocytosis, macropinocytosis and endo-lysosomal traffic Williams and Kay, 2018;Williams et al 2019;Pan et al 2018;Meena and Kimmel, 2017;Buckley et al, 20192019;Dunn et al 2018;Dinh et al 2018;Marinović et al, 2019 Cytokinesis Srivastava et al, 2016 Cell adhesion Fujimori et al 2019;Lampert et al 2017 Autophagy Mesquita et al 2017;Fischer et al 2019 Development, pattern formation Yamada and Schaap, 2019 Social evolution Ostrowski, 2019 Dictyostelium as model organism 325…”

Living on soup: macropinocytic feeding in amoebae

“…A double mutant of PI3K1 and PI3K2 grows poorly in axenic medium and when investigated in detail was found to be defective in macropinocytosis (Buczynski et al, 1997). A full analysis of the not so far been knocked out (Williams et al, 2019a).…”

“…These gain of function experiments do not replicate in axenic Dictyostelium cells, presumably because Ras is already partially activated by loss of NF1. However, expression of activated Ras in wild-type cells, with intact NF1, does stimulate macropinocytosis (Williams et al, 2019a). Active Ras forms patches in macropinosomes coincident with PIP3 (Sasaki et al, 2007, Veltman et al, 2016.…”

“…Dictyostelium has a plethora of Ras genes but the key Ras proteins for macropinocytosis appear to be RasG and RasS , Bolourani et al, 2006, Williams et al, 2019a. However, a double mutant still takes up fluid at about 35% of the parental rate, indicating that at least one more Ras protein is involved, which is likely RasB (Junemann et al, 2016).…”

“…Ras is activated by GEF and inactivated by GAP proteins. The genome encodes more than 20 RasGEFs of which GefB is implicated in macropinocytosis by null mutants having a severe defect in fluid uptake in the Ax3 genetic background and a lesser one in Ax2 (Williams et al, 2019a). GefF is implicated in macropinocytosis by an insertional mutant that also has strongly reduced fluid uptake, though the gene has by a knock-out in non-axenic cells, with intact NF1.…”