Abstract
Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by ‘macropinocytic patches’ in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.
Summary statement Dictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.
Introduction
Macropinocytosis is an ancient process used by cells to take up large amounts of fluid (King and Kay, in press; Swanson, 2008). Actin-driven protrusions are extended in a cup-shape and then close to engulf extracellular medium into an internal vesicle (Buckley and King, 2017). Macropinocytosis occurs in a variety of mammalian cell types and clinically important settings, perhaps most notably by certain cancer cells as a way of obtaining nutrients (Bloomfield and Kay, 2016; Commisso et al., 2013; Kim et al., 2018; Palm et al., 2017).
Macropinocytic, and phagocytic, cups are formed by a ring of protrusive F-actin under the plasma membrane that is distinct from other large F-actin structures, such as pseudopods. Several cellular components involved in the organisation of these cups have been identified in mammalian and Dictyostelium cells, the most important of which seem to be Ras, Rac1 and the phospholipid PIP3 (and, by extension, its regulators PI3-kinases and PTEN) (Araki et al., 2007; Bar-Sagi and Feramisco, 1986; Fujii et al., 2013; Hoeller et al., 2013; Ridley et al., 1992; Veltman et al., 2016; Yoshida et al., 2009).
The proteins organizing macropinocytic cups are better known as members of both the growth factor signalling cascade and as oncogenes or tumour suppressors. Growth factors signal through receptor tyrosine kinases (RTKs), which activate downstream effectors including Ras (Margolis and Skolnik, 1994). Active Ras in turn activates class 1 PI3-kinases, recruited to the plasma membrane by RTKs, through their Ras binding domain to make PIP3, an interaction that is critical for growth of certain tumours (Castellano et al., 2013; Gupta et al., 2007; Hu et al., 1992). PI3-kinase activation leading to macropinocytosis can also occur independently of Ras (Palm et al., 2017). Activating mutations in the RTK/Ras signalling pathway occur in nearly half of cancers, and activating mutations of the PI3-kinase pathway, mostly PI3-kinases and PTEN, in a third, although these groupings include proteins not involved in macropinocytosis (Kandoth et al., 2013; Sanchez-Vega et al., 2018). Ras proteins are especially frequently mutated with, strikingly, ∼95% of pancreatic cancers driven by K-Ras (Fernandez-Medarde and Santos, 2011; Kandoth et al., 2013; Prior et al., 2012; Waddell et al., 2015). Further, loss of the RasGAP NF1, leads to increased Ras activation and tumour development (Bollag et al., 1996; Gutmann et al., 2017).
Macropinocytosis in the model organism Dictyostelium discoideum is highly analogous to macropinocytosis in mammalian cells, however the situation is simplified as growth factor signalling and RTKs are not present. In Dictyostelium macropinocytosis is used for nutrient acquisition and accounts for the vast majority of fluid uptake, making accurate quantification simple (Hacker et al., 1997; Williams and Kay, 2018a). Additionally, both forward and reverse genetic approaches can be employed (Bloomfield et al., 2015; Paschke et al., 2018).
The Dictyostelium macropinocytic cup is formed around a template ‘macropinocytic patch’ composed of activated Ras and Rac, PIP3 (although this is chemically different to mammalian PIP3) and F-actin, with a rim of the Arp2/3 activators SCAR/WAVE and WASP (Clark et al., 2014; Hoeller et al., 2013; Veltman et al., 2016). Other known components are Coronin, the myosin-I proteins and certain formins (Brzeska et al., 2016; Hacker et al., 1997; Junemann et al., 2016). The PIP3-phosphatase PTEN is excluded from the macropinocytic patch but is present on the rest of the plasma membrane (Hoeller et al., 2013; Iijima and Devreotes, 2002).
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. PIP3 acts by recruiting PIP3-binding proteins to the plasma membrane, often through a PH-domain. A considerable number of these proteins exist, but which are important for macropinocytosis is not known (Park et al., 2008; Zhang et al., 2010). The protein kinase Akt is an oncoprotein that is a major downstream effector of PIP3 in growth factor signalling and mTORC1 activation (Dibble and Cantley, 2015; Staal et al., 1977). Continuing the analogy with growth factor signalling, we examine its role in Dictyostelium macropinocytosis.
Dictyostelium has a classical Akt homologue called PkbA with a PIP3-binding PH domain that recruits it to the plasma membrane (Meili et al., 1999; Tanaka et al., 1999). It phosphorylates target proteins at the Akt consensus sequence and the phosphorylated proteins can be recognized with standard anti-phosphopeptide antibodies (Kamimura et al., 2008; Liao et al., 2010). In addition, there is variant enzyme, PkbR1, which is constitutively targeted to the plasma membrane by lipid modification and phosphorylates an overlapping set of target proteins (Kamimura et al., 2008; Liao et al., 2010; Meili et al., 2000). PkbA and PkbR1 have a conserved activation mechanism, requiring phosphorylation by TORC2 and PDK1 protein kinases (Alessi et al., 1997; Jacinto et al., 2006; Kamimura and Devreotes, 2010; Kamimura et al., 2008; Liao et al., 2010; Sarbassov et al., 2005; Stephens et al., 1998; Stokoe et al., 1997).
We show here that the Akt protein kinases, and their activating protein kinases, are required for efficient macropinocytosis. They act downstream of PIP3 to increase macropinosome size, and hence fluid uptake, by increasing the macropinocytic patch size. We identify some of the Akt targets by phosphoproteomics. Among these is the RhoGAP GacG, which is nearly essential for fluid uptake and without which cells move rapidly and make far fewer macropinosomes.
Results
PkbA and PkbR1 are together required for efficient fluid uptake by macropinocytosis
Fluid uptake by a Dictyostelium mutant lacking all Ras-activated PI3-kinases (PI3K1-5-) is practically abolished in shaking culture (Hoeller et al., 2013). However, some mutant strains have a conditional fluid uptake defect in shaking culture, but not when attached to a surface (Novak and Titus, 1997). We therefore re-assessed fluid uptake by surface-attached PI3-kinase mutants using flow-cytometry to measure uptake of fluorescent dextran. Fluid uptake was nearly abolished in PI3K1-/2- and other multiple PI3K mutants, confirming the importance of PIP3 for macropinocytosis in our assay conditions (Figure 1A).
The Dictyostelium Akt proteins, PkbA and PkbR1, may be downstream effectors of PIP3 in macropinocytosis. In vegetative cells, PkbR1 is present on the whole membrane, including macropinocytic cups (Figure 1B, Movie 1), while PkbA is recruited specifically to macropinocytic cups (Figure 1C, Movie 2), similar to Akt in mammalian cells (Yoshida et al., 2015). Akt could therefore be an evolutionarily conserved PIP3 effector during macropinocytosis.
Single and double knockouts of PkbA and PkbR1 (Figure S1A) were made in our Ax2 background using conditions that avoid the need for axenic growth (Paschke et al., 2018). Phosphorylation of Akt substrates was largely unaffected in the single knockouts and in the PI3K1-5-strain (Figure S1B) but was nearly abolished in the PkbA-/PkbR1-strain. This is likely explained by redundancy between PkbA and PkbR1, and PIP3 independent activation of PkbR1 in the absence of PIP3 (Kamimura et al., 2008) (Figure S1C).
Fluid uptake was modestly reduced in the PkbA- and PkbR1-single mutants, in agreement with previous data for PkbA-cells (Rupper et al., 2001). However, it was effectively abolished in the double mutant, showing that PkbA and PkbR1 are together essential for effective fluid uptake (Figure 1D), and phenocopying the PI3K1-5-strain.
Since PkbA and PkbR1 are together essential for efficient fluid uptake, we asked whether their activating kinases, TORC2 and PDK1 (Figure S1C) are also required. TORC2 has four subunits: Tor, PiaA/Rictor, Rip3/Sin1 and Lst8, all of which excluding Tor can be knocked out in Dictyostelium. These mutants have decreased, but not abolished, activation of PkbA and PkbR1 (Lee et al., 2005). In contrast to previously published results, fluid uptake was decreased in TORC2 component mutants (Figure 1E), although not to the same extent as in PkbA-/PkbR1-cells (Rosel et al., 2012).
Dictyostelium has two PDK1 homologs: PdkA and PdkB. PdkA, but not PdkB, is recruited to PIP3-containing membrane regions, such as macropinosomes, in a PI3-kinase dependent fashion, although binding to PIP3 has not been shown in-vitro (Kamimura and Devreotes, 2010; Liao et al., 2010). PdkA could therefore be responsible for PIP3-dependent activation of PkbA and PkbR1, like a classical PDK1 protein, with PdkB being responsible for PIP3-independent activation of PkbR1 (Figure S1C) (Alessi et al., 1997; Kamimura et al., 2008; Stokoe et al., 1997). In agreement with this model, fluid uptake by PdkA-mutants was effectively abolished, similar to PI3K1-5- and PkbA-/PkbR1-cells, but was actually increased in PdkB-cells (Figure 1F). PIP3-independent activation of PkbR1 by PdkB therefore has no function in macropinocytosis.
These results show that PkbA and PkbR1 and their activators, PdkA and TORC2, mediate efficient macropinocytic fluid uptake in Dictyostelium. The defect in PkbA-/PkbR1-cells is as severe as in PI3K1-5-cells and thus could account for their phenotype, though additional PIP3 effectors are not excluded.
PkbA-/PkbR1-cells only have proliferation defects in axenic conditions
The fluid uptake defects observed in the PkbA-/PkbR1-cells could be due to a general defect in cell physiology. We therefore set out to investigate this.
PkbA-/PkbR1-cells do not proliferate in HL5 medium (Figure 2A), consistent with the large fluid uptake defect and a previously made mutant (Meili et al., 2000). PkbA-cells have a larger proliferation defect than their fluid uptake defect would indicate. Further investigation revealed that the rate of macropinocytosis decreases in these mutants on prolonged incubation in HL5 medium (Figures S2A&B), although the reason is unknown.
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. Poorly-macropinocytosing cells can sometimes grow and be stimulated into increased fluid uptake by medium enriched with 10% FBS (Bloomfield et al., 2015). Although growth and fluid uptake of the PkbA and PkbR1 single and double mutants could be improved by supplementation of the medium (Figure 2B), similar defects in fluid uptake were apparent (Figure 2C). This was also the case for the PI3-kinase mutants (Figure 2D). In contrast, the TORC2 component and PDK1 mutants, which can still partially activate PkbA/PkbR1 at macropinocytic cups, increase their fluid uptake in HL5 medium + 10% FBS, compared to HL5 only (Figures 2E&F). Further experiments using the PI3K1-5- and PkbA-/PkbR1-strains used HL5 medium + 10% FBS, where complications due to starvation are minimised. Further experiments using the TORC2 component and PdkA-mutants are performed in HL5 medium, as this supports their proliferation (Kamimura and Devreotes, 2010; Rosel et al., 2012).
In contrast to axenic growth, all PkbA/PkbR1 mutant strains proliferated rapidly on bacteria (Figure 2G), at similar rates to Ax2. PkbA-/PkbR1-, PI3K1-5- and Ax2 cells are a similar size when cultivated on bacteria, indicating the mutants have no abnormalities in cell cycle progression (Figure 2H). PI3K1-5- and PkbA-/PkbR1-cells are smaller than Ax2 when incubated in HL5 medium + 10% FBS, most likely due to the fluid uptake defect. Consistent with previous data, neither strain enters the developmental cycle when cultivated on bacterial lawns (Hoeller and Kay, 2007; Meili et al., 2000).
The normal growth of PkbA-/PkbR1-cells on bacteria and our inability to restore normal rates of fluid uptake to them under any nutritional condition tested indicate that they have a specific defect in macropinocytosis, rather than in general cell physiology or the regulation of macropinocytosis.
Macropinocytic patches appear to be normally organized in PkbA-/PkbR1-cells
To investigate the cause of the fluid uptake defect in PkbA-/PkbR1-cells, we examined the organisation of their macropinocytic patches. Patches of the upstream components active Ras and PIP3 were still formed in the mutant (Figure 3A) and PTEN was excluded from them (Figure 3B), as expected. F-actin (Figure 3C) and activated Rac (Figure 3D) localisation, relative to active Ras, was also unperturbed in the mutant. SCAR/WAVE was examined using basal patches of active Ras, a surrogate for macropinosomes, as the marker is weak and easily bleached (Veltman et al., 2016) (Figure 3E). SCAR/WAVE was still enriched at the patch periphery. When this was quantified, no decrease in SCAR/WAVE enrichment at the ring was observed for the PkbA-/PkbR1-cells nor, in this instance, for the PI3K1-5-, contrary to expectations (Veltman et al., 2016).
PkbA and PkbR1 increase the size of macropinocytic patches
We used microscopy to examine the rate at which macropinosomes form and their size. Macropinosomes form at a reduced rate in the PI3K1-5- and PkbA-/PkbR1-strains compared to Ax2 (Figure 4A), although this is not statistically significant and cannot account for the entire fluid uptake defect. We tested, and ruled out, the possibility that these macropinosomes were rapidly recycled to the extracellular medium by using a short uptake time (Figure S3A).
Instead, we find that PI3K1-5- and PkbA-/PkbR1-mutants form smaller macropinosomes. Macropinosome diameter at internalisation was reduced by ∼40% in both cases (Figure 4B), corresponding to a 3-4 fold decrease in macropinosome volume and thus accounting for a large proportion of the decrease in fluid uptake. A similar reduction in macropinocytic patch size was observed (Figure 4C), indicating a defect in macropinocytic patch size leads to smaller macropinosomes.
The size of particles that cells can phagocytose correlates with the size of macropinocytic patches formed (Bloomfield et al., 2015; Williams and Kay, 2018b). We measured phagocytosis of various size particles by PkbA-/PkbR1- and PI3K1-5-cells. The PkbA-/PkbR1- and PI3K1-5-mutants were less proficient than Ax2 at uptake of yeast (a comparatively large particle) (Figure S3C), while phagocytosis of smaller beads was unchanged (Figure S3D), consistent with the smaller macropinocytic patches made by these mutants.
The TORC2 component knockout and PdkA-mutants have similar phenotypes to the PI3K1-5- and PkbA-/PkbR1-cells. Macropinosome formation was reduced, but not to a statistically significant extent, in all the TORC2 component mutants, but was significantly reduced for PdkA-cells (Figure 4D). For all these mutants, macropinosome size was decreased (Figure 4E). However, while PdkA-mutants had a corresponding decrease in macropinocytic patch size, the TORC2 component mutants were largely unaffected in this regard (Figure 4F). This data is summarised in Figure 3G.
These results show that PI3K1-5- and PkbA-/PkbR1-cells make significantly smaller macropinocytic patches and macropinosomes than their parent, which (due to the cubic relation of linear dimensions and volume) leads to a much larger reduction in macropinosome volume. These results further imply that there is a local positive feedback loop in which the Akt protein kinases PkbA and PkbR1 increase the activity of upstream macropinocytosis components, thus enlarging macropinocytic patches.
Identification of PkbA/PkbR1 targets by phosphoproteomics
The postulated feedback loop between upstream macropinocytosis components and PkbA/PkbR1 might operate indirectly and by many different potential routes, but ought to be mediated by targets of both PkbA and PkbR1. We therefore used an unbiased phosphoproteomic screen to identify such targets.
We compared the phosphoproteomes of parental Ax2 cells, PkbA-, PkbR1- and PkbA-/PkbR1-mutants and PkbR1-cells treated with LY294002 to inhibit PI3-kinase and hence PkbA. Phosphopeptides were isolated from these cells as described in the methods and the relative amounts of each phosphopeptide in each sample were compared by Tandem Mass Tag mass spectrometry over three biological replicates (Data S1). The phosphopeptides were filtered by reference to the fluid uptake data obtained earlier; ≥5-fold decreased in the PkbA-/PkbR1-mutant compared to Ax2, ≥2-fold decreased in the PkbR1-+ LY294002 cells with at least a 20% decrease in either of the PkbA- and PkbR1-samples. Only phosphopeptides appearing in at least 2 of the 3 repeats were accepted. Only 38 peptides from 30 proteins met these stringent criteria.
The kinases likely to be responsible for specific phosphorylations of the filtered set of peptides were predicted using NetPhos 3.1 (Blom et al., 2004). Proteins likely to be phosphorylated by PkbA/PkbR1 are shown in Table 1, while those more likely to be phosphorylated by other kinases are in Table S1. Of the potential PkbA/PkbR1 targets, half are predicted to function in signalling, while others are predicted to be involved in endosome trafficking and digestion (Figure 5A). GO analysis of the identified proteins revealed strong enrichment for terms associated with G-protein function, intracellular transport and enzyme function, while the most enriched term was for protein complex scaffold activity (Figure 5B). These predicted functions are consistent with proteins involved in cellular feeding by macropinocytosis. Additionally, several of these proteins have previously been identified as PkbA/PkbR1 targets, validating the approach (Charest et al., 2010; Kamimura et al., 2008; Tang et al., 2011).
Since the PkbA/PkbR1-mediated increase in macropinocytic patch size most likely occurs through signalling events, we focused on the targets involved in G-protein function, protein complex scaffold activity and an identified protein kinase. A mutant of the ScaA scaffold protein was obtained from the Dictyostelium stock centre (Fey et al., 2013; Sawai et al., 2008), while knockout mutants were made of GacG, GacH, GefS and KrsB (Figure S4). We were unable to make a GxcX-mutant.
Fluid uptake of these mutants was measured in HL5 medium (Figure 5C) and HL5 medium +10% FBS (Figure 5D) and found to be decreased only in ScaA-cells (~50%) and GacG-cells (>90%, similar to PI3K1-5- and PkbA-/PkbR1-). That no defect was found for knockouts of the other proteins may be due to redundancy or compensatory elevated macropinosome formation rates that obscure a smaller macropinosome phenotype. As ScaA has previously been shown to be involved in a PkbA/PkbR1 activation feedback loop, we focused on GacG, which has a RhoGAP and a FERM domain (Charest et al., 2010).
GacG-mutants have decreased macropinocytosis and increased motility
Loss of GacG, as a RhoGAP, is expected to lead to over-activation of one or more Rho proteins. Experiments in mammalian cells have shown that Rac1, a Rho protein, must be inactivated for macropinosome internalisation, and data suggests a similar function in Dictyostelium (Dumontier et al., 2000; Fujii et al., 2013). If Rac1 inactivation does not occur, arrested macropinocytic structures can accumulate. To test if such a scenario occurs in GacG-cells, we examined macropinocytic patches using live-cell confocal microscopy. No obvious defect in macropinosome internalisation was observed, and neither was there an accumulation of macropinocytic structures. In fact, there were far fewer macropinocytic patches formed (Figure 6A). The macropinosome formation rate is also greatly reduced (Figure 6B), and those that do form are smaller (Figure 6C). Despite the fluid uptake defect, GacG-cells obtain sufficient nutrients to proliferate in HL5 medium + 10% FBS, although much slower than Ax2 cells (Figure 6D). The comparative lack of macropinocytosis is therefore not due to the cells prematurely entering the developmental cycle when in axenic conditions.
The GacG-fluid uptake defect was not rescued by overexpression of either GacG-GFP or GAP-dead GFP-GacG, which both localise evenly throughout the cytosol (not shown), while cells were highly resistant to overexpression of GFP-GacG, hampering further investigation of GacG function and localisation.
Macropinocytosis and cell movement both require the construction of large actin-driven projections by cells – cups and pseudopods – and in active cells may be in competition for limited cytoskeletal resources (Vargas et al., 2016; Veltman et al., 2014). We noticed that GacG-cells moved much more freely and faster than the parent Ax2 strain. The difference is greatest with cells grown in both HL5 medium + 10% FBS (Figure 6E), where macropinocytosis is favoured in Ax2. This difference is still present, but is reduced, when cells are grown on bacteria, where Ax2 macropinocytosis is lower (Figure 6F). The differences are also clear in cell tracks (Figures 6G&H).
GacG-cells take up much less fluid than Ax2 cells due to a decrease in macropinocytic patch (and thereby macropinosome) formation and appear to have switched cytoskeletal resources from macropinocytosis to movement, moving considerably faster in all circumstances investigated.
Discussion
In this work we show that, together, the Akt protein kinases PkbA and PkbR1 are nearly essential for fluid uptake in Dictyostelium. The defect in fluid uptake by PkbA-/PkbR1 cells is as great as that for cells lacking all Ras-activated PI3-kinases. In both mutants, this is because of a decrease in macropinosome volume due to a decrease in macropinocytic patch size. As Akt acts downstream of PIP3, lack of activation of Akt could account for the phenotype of the PI3-kinase mutants, although a role for other PIP3-binding proteins is not excluded.
PIP3 is required for effective macropinocytosis in mammalian cells, but the role of Akt is less clear (Araki et al., 1996). Inducing macropinosome formation activates Akt at macropinosomes, and this activation can be inhibited with macropinocytosis inhibitors (Chiasson-MacKenzie et al., 2018; Erami et al., 2017; Yoshida et al., 2018 preprint). However, macropinosome formation is not affected by inhibition of Akt in macrophages (Pacitto et al., 2017; Yoshida et al., 2015). In other cell-types, the opposite is true: Akt inhibition inhibits macropinocytic uptake of collagen by stellate cells (Bi et al., 2014). Akt1 deletion reduces tumour development in mice with activated Ras or PTEN deficiency, mutations which allow tumour cells to use macropinocytosis for nutrient acquisition (Chen et al., 2006; Commisso et al., 2013; Kim et al., 2018; Skeen et al., 2006). The functional relevance of Akt activation at macropinosomes remains to be fully investigated it could, in a subset of cell-types, increase macropinosome volume and fluid uptake.
Macropinocytic cups form spontaneously in the plasma membrane of Dictyostelium cells, without the need for receptor stimulation. Therefore, the role of the Akt protein kinases PkbA and PkbR1 in macropinocytosis is unlikely to be in conventional signal transduction to activate macropinocytosis. The evidence presented here shows that a major function of PkbA and PkbR1 is to make these cups larger, and so able to engulf more liquid, or in the case of phagocytosis, take up larger particles. This role, in which Akt activation leads to a local increase in activated Ras (as indicated by larger macropinocytic patches) and hence its activator, PIP3, suggests that Akt is part of a local positive feedback loop.
Our search by phosphoproteomics for targets of Akt and possible components of the postulated activating feedback loop yielded a small list of proteins. Among these we focussed on GacG, a putative RhoGAP, mutants of which have nearly abolished fluid uptake and increased movement. 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). GacG-cells are phenotypically similar to cells grown on bacteria when growing in axenic conditions, indicating GacG may somehow be required for proper axenic adaptation. It is possible that GacG might act as a switch between pseudopods and macropinosomes by suppressing pseudopod formation.
Ras, the RasGAP NF1, Ras-activated PI3-kinase, the PIP3 phosphatase PTEN and Akt are all important for macropinocytosis in Dictyostelium. In mammalian cells, Ras, PI3-kinase, PTEN and, in some instances, Akt have likewise been linked to macropinocytosis. These proteins are better known as oncogenes or tumour suppressors. Both activated Ras and PTEN deficiency stimulate macropinocytosis in mammalian cells, supporting proliferation (Commisso et al., 2013; Kim et al., 2018; Palm et al., 2017). PI3-kinase inhibition inhibits macropinocytosis and inhibition of Akt may have a similar effect in certain cell-types (Araki et al., 1996). It is possible that activating mutations in PI3-kinases and Akt may similarly support increased nutrient acquisition by macropinocytosis. If this is so, inhibiting macropinocytosis may be an effective way of inhibiting the growth of tumours activated in different components of the RTK-Ras-PI3-kinase-Akt pathway.
Materials and Methods
Dictyostelium culture conditions
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 N2 (l) and scraped out using a 16G hypodermic needle (Becton Dickinson) onto K. aerogenes and SM agar when needed. Typically cells were passaged once a week for 4-5 weeks before being refreshed. Except where indicated, we used a standardized set of mutants created in our laboratory strain of Ax2, most of which were made using transformation conditions that did not depend on axenic proliferation, reducing the risk of suppressing mutations.
Cell uptake measurements
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 1×105 cells/ml, with triplicate 50 µl samples in wells of a 96-well plate. This was incubated for 24 h at 22°C before 50 µl of the same medium containing 1 mg/ml TRITC-Dextran (155 kDa, Sigma-Aldrich) was added for 1 h unless otherwise specified. This was then thrown off, the plate washed by submersion in ice-cold KK2 buffer (16.6 mM KH2PO4, 3.8 mM K2HPO4, pH 6.1) and the cell detached using 100 µl 5 mM sodium azide dissolved in KK2MC (KK2 + 2 mM MgSO4, 100 µM CaCl2). The fluorescence of each cell in the samples was then measured by an LSR_II flow cytometer (BD Biosciences) and analysed using FlowJo.
For measurement of phagocytosis, cells were harvested from bacteria as above. In the case of 1.5 µm bead (Polysciences) uptake, the cells were diluted into KK2MC and plated as before, and given ∼30 min to settle. 1.5 µm YG beads were washed free of sodium azide and diluted into KK2MC at 2×108 beads/ml. 50 µl of this was added to each well for 20 min, after which cells were washed, detached and internalised fluorescence measured as before and analysed as described (Sattler et al., 2013). For yeast uptake, the cells were resuspended in 5 ml conical flasks at 5×106 cells/ml and shaken for ∼30 min before addition of 1×107 sonicated TRITC-yeast particles/ml. 200 µl samples were taken at 0 and 60 min, mixed with 2 µl 0.4% Trypan Blue (Sigma-Aldrich) by shaking for 3 min, washed twice in KK2 + 10 mM EDTA and the fluorescence determined on a Perkin Elmer LS 50 B fluorimeter (excitation at 544 nm, emission at 574 nm, each with a 10 nm slit).
Transformation of Dictyostelium
Vectors were made according to standard procedures, propagated using Escherichia coli XL10 and harvested using the ZR miniprep classic kit (Zymogen).
Approximately 1×106 Dictyostelium cells were harvested from growth zones on bacterial plates into 1 ml H40 buffer, pelleted by centrifugation at max speed on a benchtop centrifuge for 10 sec and resuspended in 100 µl H40 (Paschke et al., 2018). Vector was added to the cells (500 ng overexpression vector, 2 µg linearised knockout vector) and they were chilled on ice with 2 mm electroporation cuvettes (SLS). The cells were transferred to the 2 mm cuvettes then subject to square wave electroporation in a Bio-Rad GenePulser Xcell (2 × 350 Volts for 8 ms, with a 1 sec gap) and added to 2 ml KK2MC + 2 OD600 nm K. aerogenes in a 3.5 cm dish to recover for 5 h.
After recovery, for cells transformed with an overexpression vector the appropriate selection (10 µg/ml G418, although this was doubled when working with the PkbA-/PkbR1-cells due to poor marker expression, and 100 µg/ml hygromycin) was added to the dish and the dish swirled to ensure an even distribution. In the case of knockout transformations, two 50 ml falcon tubes containing 30 ml KK2MC + 2 OD600 nm K. aerogenes and selection were prepared, and the dish split between the two of them (200 µl and 1.8 ml). These were vortexed to mix and 150 µl put into each well of two 96-well plates per tube, and incubated at 22 °C. Transformants were typically obtained after ∼4 days (overexpression) and ∼6-8 days (knockout).
Screening for mutants
Confluent wells of cells resistant to drug were obtained. To screen these, the media in the well was pipetted up and down to resuspend the cells and 2 µl from the well was added to 20 µl lysis buffer with 20 µg/ml freshly added proteinase K in a 0.2 ml PCR tube (Paschke et al., 2018). The tube was vortexed to mix, then the proteinase K inactivated at 95°C for 1 min. 2 µl of DNA was added to a 25 µl PCR reaction to screen from the resistance cassette to outside the construct, giving a product that could only derive from a mutant.
Once wells containing mutants were identified, these were plated clonally onto SM plates with K. aerogenes and individual colonies were screened (colony appearance usually took 4 days), with DNA this time being prepared using a g-DNA miniprep kit (Zymogen).
To make double mutants, the resistance cassette in a single mutant strain was removed by Cre-Lox recombination: 500 ng of pDM1489 was transformed into cells, selected for, and then plated out on SM plates with K. aerogenes to remove the selective pressure. Clonal populations that had lost both resistance markers were identified; sometimes this took more than one passage of cells on bacteria.
Microscopy
Macropinosome formation rate
Cells were harvested from bacterial plates, washed free of bacteria and incubated in 2 ml of the indicated medium for 24 h at 1×105 cells per well of a 6 well plate. The medium was removed and the cells resuspended in SUM and transferred to a 2-well microscope slide (Nunc) and allowed to settle for ∼ 1 h. The medium was removed and 0.5 ml SUM containing 2 mg/ml FITC-dextran (70 kDa, Sigma-Aldrich) was added for 1 min, removed and the cells washed 2x with KK2MC, fixed using 4% paraformaldehyde for 20 min, and washed 5x with PBS (pH 5) and stored at 4°C. Z-stacks with 0.1 µm steps were taken using a Zeiss 700 series microscope and the number of dextran positive vesicles counted manually using FIJI.
Live cell microscopy
Cells were transformed with overexpression vectors as above. Transformants were washed free of bacteria and 1×105 were incubated in 2 ml medium for 24 h in a 6 well plate at 22°C. The medium was removed and replaced with SUM, and the cells transferred to 2-well microscope slides for ∼30 min before imaging using a Zeiss 700 series microscope. Movies were typically taken for 5 min with 1 frame per sec. The active Ras marker Raf1-RBD was used to observe macropinosomes and macropinocytic patches unless otherwise specified. When measuring macropinosome diameters, random cross-sections of cells were used meaning any individual diameter measured using this technique could be at any section through the macropinosome, but in the aggregate the average tends to halfway between the equator and the poles.
Motility assay
Cells were cultivated on SM agar plates in co-culture with Klebsiella aerogenes bacteria. 24 h before the assay, cells were transferred into 2 ml of either HL5 medium + 10% FBS or a K. aerogenes/KK2MC suspension (OD600 nm = 2) at 2×105 cells/ml and plated into 6-well dishes. After 24 h, cells were washed 5 times in KK2MC and resuspended in the same buffer to a final density of 1×105 cells per ml. The cells were finally seeded in a MatTek 35 mm petri dishes with a 20 mm 1.5 coverglass and allowed to attach for 20 min. The random motility of the amoebae was observed for 30 min, taking frames every 30 sec using a Zeiss 700 series microscope equipped with a 10x air objective. Automatic cell tracking was performed as described previously (Susanto et al., 2017).
Mean Generation Time determination
To measure proliferation in shaking axenic culture, cells were cultured to logarithmic phase in shaking axenic culture, diluted to 5×105 cells/ml in fresh medium and counted approximately every 12 h using a haemocytometer.
To measure axenic proliferation of surface attached cells; cells were prepared as above, then 2×104 were plated in 0.5 ml fresh medium in duplicate in multiple 24 well plates which were placed in a 22°C incubator. Approximately every 12 h, a plate was removed from the incubator and the media aspirated. The wells were washed once with buffer before staining with 0.1% crystal violet dissolved in 10% ethanol for 20 min. The free crystal violet was removed by washing three times with water, after which the crystal violet staining the cells was dissolved by addition of 0.9 ml 10% acetic acid for 20 min. The absorbance at 590 nm of the resulting solution was obtained using a Nano Drop 2000 (Thermo Scientific) and the background crystal violet staining from a set of wells with no cells was subtracted.
Finally, to measure proliferation with bacteria as the food source, cells were cultivated on SM plates in conjunction with K. aerogenes. 1×104 cells/ml from growth zones were seeded in a conical flask containing 5 ml KK2MC + K. aerogenes (at 20 OD600 nm) and the cell density counted approximately every 4 h during the daytime using a haemocytometer.
Western Blots
Cells were harvested from SM plates and washed free of bacteria. 1×106 cells were inoculated into 10 ml HL5 medium + 10% FBS in a 9 cm tissue culture plate for 24 h. They were then resuspended by pipetting, pelleted by centrifugation at 370 x g for 3 min, the supernatant discarded, and washed twice in 50 ml ice-cold KK2MC buffer. Cells were counted and transferred to a 1.5 ml eppendorf tube, spun down at max speed in a benchtop centrifuge for 10 s, the supernatant removed by aspiration, resuspended to 2×107 cells/ml in 1x sample buffer (NuPage) containing 2.5% 2-mercaptoethanol and protease and phosphatase inhibitors and incubated for 5 min at 95°C.
10 µl of the cell solutions were run out on 4-12% Bis Tris 10 well gels (Nupage) with See Blue Plus 2 used as a ladder. These were blotted onto immobilon P (Merck) membranes. This was blocked with 2% BSA (Fisher Scientific) in TBS-T for 2-3 h, then incubated with primary antibody (DM1A and 23C8D2, CST) overnight in 5% BSA in TBS-T. Membranes were rinsed twice with TBS-T and washed thrice for 10 min before addition of the secondary antibody conjugated to HRP (172-011, BioRad and Ab97051, Abcam) in 5% BSA for 2-3 h, then washed again. Antibodies were imaged with GE Healthcare detection reagent on a BioRad chemidoc using the chemidoc hi-resolution setting. Blots were stripped using restore PLUS western blot stripping buffer (Thermo-fisher) as per the manufacturers instructions before re-probing.
Cell Size
Cell size was determined using an Eclipse flow cytometer (Sony iCyt). Cells were taken directly from their growth condition/treatment and, without washing, passed through a 70 µm filter (Sysmex CellTrics), and run through the Eclipse, which measured the median diameter and volume of the cell population.
Mass Spectrometry
Sample preparation for mass spectrometry
Approximately 1-2×108 vegetative cells for each strain (Ax2, PkbA-, PkbR1-, PkbA-/PkbR1-) were harvested from cultivation on bacterial SM agar plates and washed five times in KK2MC using benchtop centrifugation at 280 x g for 3 min. The harvested cells were resuspended to 2×106 cells per ml in HL5 medium + 10% FBS, and incubated in a shaking conical flask at 22° C for 24 h.
The cells were then pelleted in a benchtop centrifuge as before, washed twice in KK2MC and resuspended to 1×107 cells per ml in KK2MC. The PkbR1-cells were split between two conical flasks, one of which had LY294002 (Cayman Chemical) added to 100 µM (to prevent PkbA recruitment to macropinosomes and activation) while the rest of the samples all had 0.2% DMSO vehicle added (Ax2 + DMSO = sample 1, PkbA-+ DMSO = sample 2, PkbR1-+ DMSO = sample 3, PkbR1-+ LY294002 = sample 4, PkbA-/PkbR1-+ DMSO = sample 5). The cells were incubated shaking at 22 °C, 180 rpm for 30 min, then mixed in a 1:1 ratio with 10% TCA, and incubated on ice for at least 30 min to lyse the cells and precipitate the protein.
The protein was pelleted in a benchtop centrifuge by spinning at 2400 x g for 10 min, then washed twice with 20 ml of ice-cold acetone under the same conditions to remove the TCA. The protein was resuspended in protein solubilisation buffer (8M Urea, 20 mM HEPES, pH 8) and the concentration of the samples was measured using a Bradford assay (BioRad) and adjusted to 2.5 mg/ml, frozen in dry ice and stored at −80° C.
Enzymatic Digestion
Following the isolation of protein, 450 µg of each sample was reduced with 5 mM DTT at 56°C for 30 min then alkylated with 10 mM iodoacetamide in the dark at room temperature for 30 min. They were then digested with mass spectrometry grade Lys-C (Promega) at a protein: Lys-C ratio of 150: 1 (w/w) for 4 h at 25°C. Next, the samples were diluted from 8 M to 1.5 M urea using 20 mM HEPES (pH 8.5) and digested at 30°C overnight with trypsin (Promega) at a 75: 1 (w/w) protein: trypsin ratio. Digestion was stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 1%. Any precipitates were removed by centrifugation at 13000 x g for 15 min. The supernatants were desalted using homemade C18 stage tips containing 3M Empore extraction disks (Sigma-Aldrich) and 8 mg of poros R3 (Applied Biosystems) resin. Bound peptides were eluted with 30-80% acetonitrile (MeCN) in 0.1% TFA and lyophilized.
TMT peptide labelling
The lyophilized peptides from each sample were resuspended in 75 µl of 3% MeCN. The peptide concentrations were determined by Pierce Quantitative Colorimetric Peptide assay (Thermo Scientific) according to the manufacturers’ instructions, except the absorbance was measured by Nanodrop Spectrophotometers (Thermo Scientific) at 480 nm. 0.8 mg of each TMT 10plex reagent (Thermo Scientific) was reconstituted in 41 µl anhydrous MeCN. The peptides from each of the samples were labelled with a distinct TMT tag in 170 mM triethylammonium bicarbonate for 1 h at room temperature (r.t.). The labelling reaction was stopped by incubation with 8 µl 5% hydroxylamine for 15 min. The labeled peptides from each repeat were combined into a single sample and partially dried to remove MeCN in a SpeedVac (Thermo Scientific). After this, the sample was desalted as before and the eluted peptides were lyophilized.
Phosphopeptide enrichment
The lyophilized labeled peptides were resuspended in 1.2 ml of 50% MeCN, 2 M lactic acid (loading buffer) in an Eppendorf tube and incubated with 20 mg TiO2 beads (Titansphere, GL Sciences, Japan) at room temperature for 1 h. For the second round of enrichment, the beads slurry was spun down by centrifugation, and the supernatant was transferred to another Eppendorf tube. The supernatant was incubated with 15 mg of TiO2 beads and the beads slurry was spun down again as before. The beads were transferred to homemade C18 stage tips, washed in the tip twice with the loading buffer and once with 50% MeCN, 0.1% TFA. Phosphopeptides were eluted sequentially with 50 mM K2HPO4 (pH 10) followed by 50 mM K2HPO4, 50% MeCN (pH 10) and 50% MeCN, 0.1% TFA. The eluates were partially dried in a SpeedVac after acidification and were desalted as described above.
Basic pH Reverse-Phase HPLC fractionation
The enriched phosphopeptides were subject to off-line high performance liquid chromatography (HPLC) fractionation, using an XBridge BEH130 C18, 5 µm, 2.1 mm x 150 mm (Waters) column with an XBridge BEH C18 5 µm Van Guard cartridge, connected to an Ultimate 3000 Nano/Capillary LC System (Dionex). Phosphoeptides were resolubilised in solvent A (5% MeCN, 95% 10 mM ammonium bicarbonate, pH 8) and separated with a gradient of 1-90% solvent B (90% MeCN, 10% 10 mM ammonium bicarbonate, pH 8) over 60 min at a flow rate of 250 µl/min. A total of 58 fractions were collected. They were combined into 20 fractions and lyophilized.
LC MS/MS
The fractionated phosphopeptides were analysed by LC MSMS using a fully automated Ultimate 3000 RSLC nano System (Thermo Scientific) fitted with a 100 µm x 2 cm PepMap100 C18 nano trap column and a 75 µm × 25 cm reverse phase C18 nano column (Aclaim PepMap, Thermo Scientific). Lyophilised phosphopeptides were dissolved in solvent A (2% MeCN, 0.1% formic acid) and eluted with a linear gradient from 4 to 50% solvent B (80% MeCN, 0.1% formic acid) over 110 min with a flow rate of 300 nl/min. The outlet of the nano column was directly interfaced via a nanospray ion source to a Q Exactive Plus mass spectrometer (Thermo Scientific). The mass spectrometer was operated in standard data dependent mode, performing a MS full-scan in the m/z range of 350-1600, with a resolution of 140000. This was followed by MS2 acquisitions of the 15 most intense ions with a resolution of 35000 and NCE of 33%. MS target values of 3e6 and MS2 target values of 1e5 were used. The isolation window of precursor was set at 1.2 Da and sequenced peptides were excluded for 40 sec.
Data analysis
The acquired MSMS raw files from the analysis above were processed using Proteome Discoverer (version 2.1, Thermo Scientific). MSMS spectra were searched against the January 2016 Dictyostelium discoideum UniProt FASTA database, using Mascot (version 2.4, Matrix Science) search engine. Cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation, N-terminal acetylation (protein), phosphorylation (STY) and TMT6plex (peptide N-terminus and Lysine) were selected as variable modifications. Other parameters were set to default values. The abundance values of TMT reporter ions were normalized to total peptide amount. Two output files were generated; one listing the proteins identified and one for peptides. Only peptides that were identified with a high confidence (False discovery rate-FDR-of <1%) based on a target decoy approach were included in these results. The protein output file was filtered to only contain proteins which passed the threshold FDR of 1% and exported as an excel file. The peptides table was filtered for phosphopeptides with quantitative values and exported as an excel file.
The phosphopeptides were filtered by reference to the fluid uptake data obtained earlier: ≥5-fold decreased in the PkbA-/PkbR1-mutant compared to Ax2, ≥2-fold decreased in the PkbR1-+ LY294002 cells and have at least a 20% decrease in one of the PkbA-and PkbR1-samples. Only phosphopeptides meeting these criteria in at least 2 of the 3 repeats were accepted. The likely kinase responsible for the phosphorylation was then predicted using NetPhos 3.1.
Quantification and statistical analysis
Statistical analysis was performed using Graphpad prism software, which was used to generate the presented graphs. When there were more than two samples to compare, one-way anova was performed before any further statistical tests. Unless otherwise specified an unpaired t-test was performed and p values ≤0.01 are marked with **, those ≤0.05 * and those ≤0.1 are indicated in text. Unless otherwise stated the mean is plotted with the error bars showing the SEM.
Competing interests
The authors declare no competing interests.
Funding
We thank the Medical Research Council UK for core funding (U105115237) to RRK and Astra Zeneca for blue sky funding.
Data availability
The phosphoproteomic data set generated is available in Data S1.
Acknowledgements
The authors thank the Kay lab for their input into this project and the LMB microscopy and flow cytometry facilities for excellent scientific and technical support. We are also very grateful to Douwe Veltman for modifying his membrane finder software, and Peter Devreotes for providing the PDK mutants.
Footnotes
↵2 thomasw{at}mrc-lmb.cam.ac.uk