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The physiological regulation of macropinocytosis during Dictyostelium growth and development - PubMed

  • ️Mon Jan 01 2018

The physiological regulation of macropinocytosis during Dictyostelium growth and development

Thomas D Williams et al. J Cell Sci. 2018.

Abstract

Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium 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. Dictyostelium 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 Dictyostelium cells, regulated by environmental conditions that are identified here.This article has an associated First Person interview with the first author of the paper.

Keywords: Dictyostelium; Endocytosis; Flow cytometry; Macropinocytosis.

© 2018. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.

Fluid uptake measurement by high-throughput flow cytometry. (A) Sodium azide causes efficient detachment of cells in 96-well plates. Attached cells were incubated with sodium azide for 5 min and the proportion remaining attached was measured through Crystal Violet staining (Bloomfield et al., 2015). (B) Sodium azide prevents significant exocytosis of TRITC–dextran for at least 2–3 h. Cells, loaded with dextran, were washed and incubated in 5 mM sodium azide and intracellular fluorescence was measured by flow cytometry. (C) Representative dot-plots showing forward and side scatter for 1 μm beads, bacteria, yeast and Dictyostelium cells. Dictyostelium are easily distinguished from bacteria, beads and background particles by gating, but cannot be separated fully from yeast particles. (D) Representative histograms showing the internalised TRITC–dextran of individual cells within a population over time. Axenically grown Ax2 cells were incubated in shaking suspension with TRITC–dextran for up to 2 h and analysed by flow cytometry. TRITC–dextran accumulates in every cell, although there is a lagging tail of cells with lower fluid uptake. (E) Fluid uptake timecourse of Ax2 cells in a 96-well plate. TRITC–dextran accumulates linearly for the first 60–90 min, then plateaus as it begins to be exocytosed. (F) A comparison of the fluid uptake by cells in this study with previously published values. All error bars show s.e.m.; n=3 in all experiments.

Fig. 2.
Fig. 2.

Cells adapt to growth on liquid media by increasing their rates of fluid uptake and macropinocytosis. (A) Macropinocytosis increases when cells grown on bacteria are transferred to liquid medium. Fluid uptake was either measured immediately after harvesting cells from bacteria (control) or after 24 h in the indicated media (n=3). Conditions were compared against the control by unpaired t-test and all had P<0.02. (B) Kinetics of the increase in fluid uptake by Ax2 cells during adaptation to nutrient-containing media (n=3). The cell density is too low for multicellular development to be induced when in KK2MC buffer. (C) The rate of macropinosome formation increases in Ax2 cells adapted to nutrient-containing media. Macropinosome formation was measured by microscopy of cells fixed after a 1 min pulse with FITC–dextran (n=6, P<0.0001 for both conditions compared to the control by unpaired t-test). (D) The size of macropinosomes increases in Ax2 cells adapted to nutrient-containing media. The maximum diameter of macropinosomes at the moment of closure was measured in the mid-section of cells by using the PIP3 reporter PkgE-PH–mCherry on three separate days (P<0.0001 for both conditions compared to the control by unpaired t-test). (E) Macropinosome formation increases in DdB cells adapted to HL5 fortified with 10% FCS (n=6, P=0.057 unpaired t-test, compared against the control). (F) Macropinosome size does not increase in DdB cells adapted to liquid media (cells in HL5 had a slight decrease in size compared to the control, whereas cells in HL5+10% FCS showed no difference in size: P=0.55 by unpaired t-test). Cells were imaged on three separate days. Ax2 is a standard laboratory strain able to grow in HL5 medium due to the absence of an NF1 gene. DdB is its non-axenic parent with an intact NF1 gene. Cells were grown on bacteria, washed and then transferred to the indicated media. Excluding panel B, the control measurements were made with cells freshly harvested from bacteria and the other measurements were made after 24 h incubation in the indicated media. Fluid uptake and other measurements were made as described in the Materials and Methods. Error bars show the s.e.m.

Fig. 3.
Fig. 3.

Macropinocytosis upregulation can be induced by a minimal medium containing glucose, arginine, lysine and glutamate. (A) The defined medium SIH efficiently induces upregulation of macropinocytosis in cells transferred from bacteria. Fluid uptake in the complex HL5 medium is also shown for comparison (n=3). (B) Broad dissection of SIH medium shows that the amino acids and glucose are responsible for its ability to stimulate macropinocytosis upregulation (n=7, P=0.7 by unpaired t-test between SIH and SIH glucose+amino acids). (C) Detailed dissection of SIH medium shows that arginine, glutamate and lysine (R, E and K) are needed for efficient upregulation of macropinocytosis (n=5, P=0.0004 for SIH compared to SIH−R/E/K and 0.5 for SIH compared to SIH−R/E/K+R/E/K by unpaired t-tests). (D) A minimal medium containing only the arginine, glutamate, lysine and glucose in SIH (SUM) gives efficient upregulation of macropinocytosis. The kinetics of upregulation induced by SUM, SIH glucose and amino acids and SIH are compared (n=3). Ax2 cells grown on bacteria were washed free of bacteria and transferred to the indicated media for 24 h, unless indicated otherwise, and then fluid uptake measured by flow cytometry as described in the Materials and Methods. Error bars show the s.e.m.

Fig. 4.
Fig. 4.

Evidence that macropinocytosis upregulation depends on macropinocytosis. To test whether macropinocytosis upregulation depends on macropinocytosis, inhibitors with differing targets (see Table 1) were used to inhibit macropinocytosis during the upregulation period. The inhibitor was then washed away and the degree of upregulation determined by measuring fluid uptake compared to untreated controls (‘raw’ curves). To control for long-term effects of the inhibitors, cells with fully upregulated macropinocytosis were treated in parallel and the results corrected accordingly (‘corrected’ curves; see

Fig. S4

). Inhibitors used and their nominal targets were: (A) LY29004 (PI3K, n=3); (B) TGX221 (PI3K, n=4); (C) CK666 (Arp2/3 complex, n=4); (D) EHT1864 (Rac, n=4); (E) Torin 1 (Tor, n=3); and (F) rapamycin (TORC1, n=3). Ax2 cells, harvested from bacteria, were incubated in HL5 in 96-well plates with the inhibitors for 10 h, then the inhibitors were washed away by dunk-banging and the cells allowed to recover for 10 min before the fluid uptake was measured over 1 h using the high-throughput flow cytometry assay. To correct for deleterious effects of the inhibitors, control Ax2 cells grown in HL5 (with maximally upregulated macropinocytosis) were similarly treated with inhibitors for 10 h and their fluid uptake compared to untreated controls to give the correction factor as Uptake (drug-treated control cells)/Uptake (vehicle-treated control cells), by which the raw data was multiplied to give the corrected curves. Error bars show the s.e.m.

Fig. 5.
Fig. 5.

Long-term regulation of macropinocytosis by bacteria and their product, folate. (A) Bacteria inhibit the upregulation of macropinocytosis in cells transferred to HL5 medium. Ax2 cells transferred from bacteria (low macropinocytosis) to HL5 upregulate macropinocytosis, but this is blocked by addition of Ka bacteria (2 OD600 nm) to the HL5 (n=6). (B) Bacteria induce downregulation of macropinocytosis by cells taken from HL5 medium. Ax2 cells transferred from HL5 medium (high macropinocytosis) to KK2MC buffer maintain their rate of macropinocytosis (the cell density is too low for development), but the addition of 2 OD600 nm Ka bacteria induces downregulation (n=6). (C) Folate delays the upregulation of macropinocytosis in cells transferred to HL5 medium. Ax2 cells transferred from bacteria (low macropinocytosis) to HL5 medium upregulate macropinocytosis, but this is delayed upon addition of 500 µM folate (n=6, P=0.025 by unpaired t-test at 6 h upregulation). (D) The folate receptor (fAR1) mediates the inhibitory effect of folate on macropinocytosis upregulation. Wild-type Ax2 cells and a null mutant for the folate receptor (fAR1−) were transferred from bacteria (low macropinocytosis) to HL5 medium with or without 500 µM folate and macropinocytosis measured after 6 h (n=5, comparing the values of the folate treated cells, P=0.0004 by unpaired t-test). (E) The heterotrimeric G-protein cognate to the folate receptor mediates the inhibitory effect of folate on macropinocytosis upregulation. Wild-type Ax2 cells and null mutants for Gα4 (gpaD−) and Gβ (gpbA−) were transferred from bacteria (low macropinocytosis) to HL5 medium with or without 500 µM folate and macropinocytosis was measured after 6 h (n=5, comparing the values of the folate treated cells to the untreated cells, P=0.18 and 0.07 by unpaired t-test for Gα4− and Gβ−, respectively). (F) The MAP-kinase ErkB, a downstream effector of the folate receptor mediates the inhibitory effect of folate on macropinocytosis upregulation. Wild-type Ax2 cells and null mutants for ErkB (erkB−) were transferred from bacteria (low macropinocytosis) to HL5 medium with or without 500 µM folate, and macropinocytosis was measured after 6 h (n=3, comparing the values of the folate-treated cells to the untreated cells, P=0.65 by unpaired t-test). Fluid uptake was measured with the high-throughput flow cytometry. Error bars are the s.e.m.

Fig. 6.
Fig. 6.

Macropinocytosis is downregulated by developmental signalling that likely acts through PKA. (A) Macropinocytosis is downregulated during development. Ax2 cells grown in HL5 (high macropinocytosis) were washed free of nutrients and allowed to develop in standard conditions: shaken in suspension and pulsed with cyclic AMP every 6 min after the first hour (n=5). (B) Downregulation of macropinocytosis depends on the cell density. Axenically growing cells were allowed to settle at high (50,000 cells well−1) and low (5000 cells well−1) density in 96-well plates, washed free of nutrient media and incubated in KK2MC buffer for the indicated times before fluid uptake was determined as described in the Materials and Methods (n=6). (C) Downregulation of cells at low density is induced by conditioned medium. Conditioned KK2MC buffer (CM) prepared by shaking starving cells at high density for 8 h was tested for its ability to induce downregulation of macropinocytosis in Ax2 cells. The CM was size-fractionated or heat-treated at 75°C for 30 min to further define the properties of the secreted product responsible for downregulation (n=3, P<0.0001 for CM and CM >30 kDa compared to KK2MC buffer by unpaired t-test, and 0.77 for heat-treated CM). (D) Downregulation of macropinocytosis in cells at low density incubated for 24 h with 8-Br-cAMP (which activates PKA) (n=5). (E) Mutations giving elevated intracellular cyclic AMP levels bypass the need for developmental signalling to downregulate macropinocytosis. regA− and rdeA− cells have elevated intracellular cyclic AMP due to reduced breakdown, and downregulate macropinocytosis when incubated in KK2MC buffer for 24 h, unlike Ax2 (n=5, for unpaired t-tests between HL5 and KK2MC buffer P=0.0003 for rdeA− and regA− and 0.53 for Ax2). (F) Macropinocytosis is not downregulated in a mutant lacking PKA activity (pkaC−), even when incubated in CM or at high density in KK2MC buffer (n=6). Error bars show the s.e.m.

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