The physiological regulation of macropinocytosis during Dictyostelium growth and development

Abstract

Macropinocytosis is a conserved endocytic process used by amoebae for feeding on liquid medium. To further as a model for macropinocytosis, we developed a high-throughput flow cytometry assay to measure macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. has two feeding states: phagocytic and macropinocytic. When cells are switched from phagocytic growth on bacteria to liquid media, the rate of macropinocytosis slowly increases, due to increased size and frequency of macropinosomes. Upregulation is triggered by a minimal medium containing three amino acids plus glucose and likely depends on macropinocytosis itself. The presence of bacteria suppresses macropinocytosis while their product, folate, partially suppresses upregulation of macropinocytosis. Starvation, which initiates development, does not of itself suppress macropinocytosis: this can continue in isolated cells, but is shut down by a conditioned-medium factor or activation of PKA signalling. Thus macropinocytosis is a facultative ability of cells, regulated by environmental conditions that are identified here.This article has an associated First Person interview with the first author of the paper.

Akt kinases are required for efficient feeding by macropinocytosis inDictyostelium

“…The rate of macropinocytosis by Ax2 cells increases massively in the first 10 h after transfer to liquid medium, and this increase likely depends on nutrient sensing within the macropinocytic pathway (Williams and Kay, 2018b). If insufficient nutrition is obtained, upregulation does not occur.…”

“…Macropinocytic cups and pseudopods are both substantial F-actin structures and may compete for the same cytoskeletal resources in active cells. In Dictyostelium , cells grown in liquid medium produce many macropinosomes but move poorly, while it is the reverse for cells grown on bacteria, as well as cells undergoing development (Fisher et al, 1989; Veltman et al, 2014; Williams and Kay, 2018b). This competition between macropinocytosis and movement is also observed in dendritic cells (Vargas et al, 2016).…”

“…Dictyostelium discoideum Ax2 cells and derivatives were used in this study (See supplementary table 1) and were cultivated on Klebsiella aerogenes bacteria on SM plates at 22°C unless otherwise stated. Nutrient media (HL5 (Formedium, UK), HL5 + 10% FBS and SUM (Williams and Kay, 2018b)) were supplemented with Dihydrostreptomycin (100 µg/ml), Ampicillin (100 µg/ml) and Kanamycin (50 µg/ml). Strains were stored frozen in Horse Serum + 7.5% DMSO in N 2 (l) and scraped out using a 16G hypodermic needle (Becton Dickinson) onto K. aerogenes and SM agar when needed.…”

“…All cell uptake measurements were performed as described (Williams and Kay, 2018a; Williams and Kay, 2018b). Briefly, cells were cultivated on SM agar with K. aerogenes bacteria, harvested, washed free of bacteria and inoculated in the indicated nutrient medium to 1×10 5 cells/ml, with triplicate 50 µl samples in wells of a 96-well plate.…”

“…PIP3 is vital for efficient macropinocytosis, in both amoebae and mammalian cells (Araki et al, 1996; Hoeller et al, 2013; Williams and Kay, 2018b). However, despite its importance, its function in macropinocytosis is unknown.…”