Author response: A plasma membrane template for macropinocytic cups (2016)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery. https://doi.org/10.7554/eLife.20085.001 eLife digest Cells can use a process known as macropinocytosis to take up fluid from their surroundings. This process plays an important role in many situations. For example, it allows human immune cells to sample their environment to search for harmful microbes and viruses and helps cancer cells to collect more nutrients so that they can grow more rapidly. During macropinocytosis, a protein called actin – which provides structural support to cells – drives the formation of cup-shaped structures from the membrane that surrounds the cell. Several signaling molecules control when and where the “cups” form, but it was not known exactly how the different types of molecules work together. Here Veltman et al. used a technique called lattice light sheet microscopy to investigate how the macropinocytic cups form in a single-celled amoeba known as Dictyostelium. The experiments revealed that to make a cup, the actin first arranges to form a ring. The ring copies a template in the membrane, which consists of high concentrations of signaling molecules, and then extends outward to form a hollow cup by which fluid is taken up. The most important signaling molecule identified in these patches of membrane is a protein called Ras, which is mutated and hyperactive in many different types of cancer. In Dictyostelium cells that have a genetic mutation that makes Ras more active, the patches of signaling molecules and macropinocytic cups were larger than in normal cells. The findings of Veltman et al. provide new details about how cells engulf fluids from their surroundings. The next steps will be to investigate how the signaling molecules form patches in the first place, and how they attract actin molecules. Also, more research is necessary to find out whether all cells take up fluid in a similar way or if other methods have evolved in mammalian cells. https://doi.org/10.7554/eLife.20085.002 Introduction Macropinocytosis provides cells with an efficient way of taking up large volumes of medium into intracellular vesicles, from which they can extract nutrients, antigens and other useful molecules (Bloomfield and Kay, 2016; Egami et al., 2014; Maniak, 2001; Swanson, 2008; Swanson and Watts, 1995). It is an ancient process, used for feeding by amoebae (Hacker et al., 1997; Thilo and Vogel, 1980), but one that is important for a wide spectrum of human biology, including uptake of drugs, and large-scale sampling of extracellular medium for antigens by immune cells. It has also been hijacked by pathogens as a major route of entry (Mercer and Helenius, 2012). Recent data suggest that macropinocytosis is a principal and widely used method for sustaining the excessive metabolic demands of cancer cells (Commisso et al., 2013; Kamphorst et al., 2015) and may be implicated in the spread of neurodegenerative disease within the brain (Münch et al., 2011). Considering its biological importance, macropinocytosis is not well understood. Macropinosomes form from cup-shaped extensions of the plasma membrane, often known as circular ruffles, which are extended by actin polymerisation. The leading rims of these ruffles must be driven outwards to enclose liquid – often for a very significant fraction of the cell’s diameter – but the base must be held static. The resulting cups can be several microns in diameter, and eventually close by constriction of their rim, with membrane fusion producing an endocytic vesicle. Here we address a critical and mysterious question about this process - how do cells organize actin to polymerize in a ring and so form the walls of the cup? In the closely related process of phagocytosis, in which solid particles are taken up, it is proposed that cup formation is guided by engaging receptors with the particle to be engulfed in a zippering process (Freeman and Grinstein, 2014; Griffin et al., 1975). However, macropinosomes take up fluid and so cannot use a particle as a template in this way. Nor is there any known equivalent in macropinocytosis of the coat that organises clathrin-mediated endocytosis. Thus it appears that macropinocytic cups must form by a self-organizing process within the actin polymerization machinery and its regulators. The dynamic actin that polymerises around macropinocytic cups is probably initiated by a number of nucleators, including both formins, such as ForG, which is needed for the basal part of phagocytic cups in Dictyostelium (Junemann et al., 2016), and the Arp2/3 complex (Insall et al., 2001), which produces dendritic structures (Pollard and Borisy, 2003), like the actin that drives pseudopods. Assembly of Arp2/3 based actin is controlled by the WASP family of nucleation promoting factors; the two family members that act at the plasma membrane are WASP and SCAR/WAVE (hereafter called SCAR). WASP is important for actin polymerisation during clathrin-mediated endocytosis (Taylor et al., 2011), and SCAR, acting in a five-membered complex (Eden et al., 2002), for the formation of pseudopods (Seastone et al., 2001; Veltman et al., 2012). It is not known which is responsible for macropinocytosis. Ras and phosphoinositide signalling help organize the cytoskeleton for macropinocytosis and phagocytosis (Bar-Sagi and Feramisco, 1986; Bloomfield and Kay, 2016; Bohdanowicz and Grinstein, 2013; Rodriguez-Viciana et al., 1997; Swanson, 2014). There is evidence that Ras activity stimulates macropinocytosis in both mammalian and Dictyostelium cells, and macropinocytic cups are associated with an intense domain, or ‘patch’, of PIP3 (Araki et al., 2007; Parent et al., 1998; Yoshida et al., 2009), which is essential for their function (Araki et al., 1996; Buczynski et al., 1997; Hoeller et al., 2013; Zhou et al., 1998). In macrophages, which often evolve macropinocytic cups from linear ruffles, it has been suggested that ruffle circularisation creates a diffusion barrier in the membrane leading to intensified PIP3 signalling and a domain of PIP3 in the centre of the circular ruffle (Welliver et al., 2011). This domain then drives the further progression of the macropinocytic cup. In axenic strains of Dictyostelium, which grow efficiently in liquid medium, macropinocytosis is massively up-regulated due to mutation of the RasGAP neurofibromatosis-1 (NF1) (Bloomfield et al., 2015; Hacker et al., 1997; Kayman and Clarke, 1983). These strains are thus an excellent starting point for research into the organising principles behind macropinocytic cup formation. Here we examine macropinocytosis with unprecedented 3D detail using lattice light sheet microscopy, and map the spatial and temporal control of actin regulators such as SCAR and WASP with respect to signalling molecules including PIP3 and active Ras. This leads us to propose a new and general hypothesis for the formation of cups from the plasma membrane. Results The origins of macropinosomes in axenic strains of Dictyostelium To determine whether macropinosomes form in Dictyostelium by circularization of linear ruffles, as reported for macrophages (Welliver and Swanson, 2012), we used lattice light sheet microscopy (Chen et al., 2014), which allows unparalleled high-resolution imaging of light-sensitive and dynamic cells over prolonged periods. In axenic cells expressing an F-actin reporter, three types of large F-actin structure are routinely detected: macropinocytic cups, which predominate, pseudopods and basal waves. 3D movies show that the majority of macropinocytic cups initiate by splitting of existing ones (62%; n = 152, Figure 1E). Splitting occurs by a variety of routes, including: simple division in the middle; detachment of a small ruffle that grows into a new macropinocytic cup (Figure 1A, Video 1); and abortive fragmentation of a parental macropinocytic cup into multiple daughter cups. We examined a reporter for active Rac in some of these movies and found that it is spatiotemporally associated tightly with F-actin in this morphological process (Figure 1B, Video 2). Figure 1 Download asset Open asset Life histories of macropinocytosis, pseudopods and basal waves. Vegetative axenic cells of strain Ax2 were followed in 3D time-lapse movies made by lattice light sheet microscopy. Images show maximum intensity projections. Cells express a marker for F-actin (LimEΔcoil-RFP) unless otherwise indicated. Numbers indicate time in seconds. (A) A new macropinocytic cup formed by splitting (arrow marks nascent daughter macropinocytic cup). (B) Close-up of a large macropinocytic cup, viewed en-face, that fragments and forms multiple smaller macropinocytic cups (arrowed). (C) Origin of a cluster of macropinocytic cups from a small basal F-actin structure (arrow). (D) Detail of a cell initiating a de novo dorsal macropinocytic cup (ie not in contact with substratum). (E) Table of macropinocytic cup origins. (F) Close-up of macropinocytic cup closure, viewed en face. (G) Growth of a pseudopod from a small F-actin structure close to the substratum (arrow). (H) Growth of a basal F-actin wave from a small F-actin punctum (arrow), to eventually encompass the entire basal surface of the cell. See also supplementary movie 1-7 for full movies of panel A, B, C, D, F, G and H respectively. https://doi.org/10.7554/eLife.20085.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Macropinosomes are generated by splitting. A vegetative Ax2 cell expressing an F-actin marker imaged using lattice light sheet microscopy and viewed from three perpendicular angles. https://doi.org/10.7554/eLife.20085.004 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Macropinocytic cups are generated by splitting. Detail of a vegetative Ax2 cell expressing markers for active Rac and F-actin, imaged using lattice light sheet microscopy and viewed from three perpendicular angles. https://doi.org/10.7554/eLife.20085.005 The remaining macropinocytic cups form de novo, expanding from places where no previous F-actin activity was detected. In more than 90% of cases the initiation is close to the base of the cell, even though most mature macropinocytic cups are present on the top, and are commonly described as 'crowns'. In the example illustrated in Figure 1C (Video 3), the parental ruffle first emerges close to the substratum (t = 0, white arrow) and cannot unequivocally be classified as either pseudopod or circular ruffle. The ruffle then quickly sweeps to the top of the cell, during which time it grows in size and splits several times, to produce multiple full-grown macropinocytic cups. As with splitting macropinocytic cups, the F-actin in de novo macropinosome cups is closely associated with signalling molecules, as illustrated by active Ras in Figure 1D (Video 4) and discussed later. Circular ruffles can persist on the cell surface for prolonged periods before either closing successfully or regressing back into the cell body. Closure of the cup can be quite abrupt and often appears to involve the inward collapse of the rim (Figure 1F, Video 5) (Swanson et al., 1999). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Macropinocytic ruffles often initiate on the substratum. A vegetative Ax2 cell expressing an F-actin marker imaged using lattice light sheet microscopy and viewed from three perpendicular angles. https://doi.org/10.7554/eLife.20085.006 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg A small fraction of de novo macropinocytic cups are initiated off- substratum. Detail of a vegetative Ax2 cell expressing markers for active Ras and F-actin, imaged using lattice light sheet microscopy and viewed from three perpendicular angles. https://doi.org/10.7554/eLife.20085.007 Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Closure of a macropinocytic cup. A vegetative Ax2 cell expressing an F-actin marker imaged using lattice light sheet microscopy and viewed from two perpendicular angles. https://doi.org/10.7554/eLife.20085.008 The other large F-actin projections in growing Dictyostelium cells are pseudopods. These are distinguished from macropinocytic circular ruffles by their shape, which is convex instead of concave. De novo pseudopods also initiate close to the substratum (Figure 1G, white arrow and Video 6) and expand steadily to their full size. Pseudopods are surprisingly rare in growing axenic cells, accounting for less than 5% of all large F-actin structures. Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Pseudopods are distinct from macropinocytic ruffles. A vegetative Ax2 cell expressing an F-actin marker imaged using lattice light sheet microscopy and viewed from three perpendicular angles. A pseudopod is initiated on the right hand side of the cell at t = 1 min. https://doi.org/10.7554/eLife.20085.009 Finally, we could follow the enigmatic actin waves that move across the basal surface of vegetative cells (Bretschneider et al., 2004, 2009; Gerisch, 2010). These waves also generally originate from existing ruffles by splitting. When the splitting ruffle in Figure 1H (Video 7) contacts the substratum, it initiates an actin wave that spreads across the entire footprint of the cell, becoming so dominant that other large F-actin structures are suppressed and flattening the cell into a smooth bell-shape. Video 7 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Basal F-actin wave that originates by splitting from a nascent macropinosome. A vegetative Ax2 cell expressing an F-actin marker imaged using lattice light sheet microscopy and viewed from three perpendicular angles. Image jitter in this movie was due to technical issues with the microscope's Z-drive). https://doi.org/10.7554/eLife.20085.010 These observations show that the large macropinocytic cups of axenic Dictyostelium cells generally form by splitting or by expanding de novo from a small focus, as in fibroblasts (Bernitt et al., 2015), rather than by circularization of linear ruffles (Welliver and Swanson, 2012). The smaller macropinocytic cups of wild-type cells (see later) also more normally form de novo or by splitting, rather than by circularization. PIP3, Ras and SCAR are required for normal fluid phase uptake We tested the involvement of PIP3 and Ras signalling in macropinocytosis using an isogenic set of mutants in which we measured both fluid uptake and growth in liquid medium (Supplementary material, Table 1). Either increased or decreased PIP3 levels (PTEN and PI3-kinase mutants) are deleterious to fluid uptake and growth in liquid medium, as expected from earlier work (Clark et al., 2014; Hoeller and Kay, 2007). Two independent RasG- mutants are substantially impaired in growth in liquid medium, as previously described, but contrary to the earlier report (Khosla et al., 2000), both are also defective in fluid uptake. Compensation by other Ras proteins and genetic background differences may account for the discrepancy (Bloomfield et al., 2008; Bolourani et al., 2010). RasC null cells have no growth defect and a lesser defect in fluid uptake, while RasS (not tested here) may also contribute to macropinocytosis (Chubb et al., 2000). Notably, we confirm that the Arp2/3 activator, SCAR, is required for efficient fluid uptake (Seastone et al., 2001). Table 1 Growth and fluid uptake by mutants. https://doi.org/10.7554/eLife.20085.011 StrainMutated proteinGenotypeMGT±SEM (hr)Fluid uptake ± SEM (nl/10^six cells/h)Ax2parental9.26 ± 0.26 (26) 114.7 ± 10.8 (9) HM1505RasCrasC- 9.27 ± 0.26 (7) 84.3 ± 6.3 (3) HM1497RasGrasG- 27.6 ± 2.9 (7) 55.3 ± 8.4 (3) HM1514RasGrasG- 19.1 ± 0.24 (3) 67.6 ± 1.9 (3) HM1200PI3K1-5pikA-, pikB-, pikC-, pikF-, pikG- 103 ± 1.1 (3)*9.0 ± 0.6 (3) HM1289PTENptenA- 19.0 ± 1.4 (9) 12.4 ± 0.6 (3) HM1809SCARscrA- 27.8 ± 2.4 (6) 56.8 ± 9.7 (3) HM1818SCARscrA- 38.4 ± 4.6 (10) 19.7 ± 5.0 (3) * taken from Hoeller et al. (2013). A ring of active SCAR forms around PIP3 domains at the rim of macropinocytic cups The SCAR complex is mostly cytosolic and basally inactive, but when recruited to the plasma membrane it causes actin polymerization through the Arp2/3 complex (Steffen et al., 2004; Ura et al., 2012). A GFP reporter tagged at the HSPC300 subunit accumulates at sites of actin polymerization (Veltman et al., 2012). To confirm that this accumulation signifies the presence of activated SCAR complex, we correlated the signal with the expansion of pseudopods, using this as a proxy for actin polymerization. The results clearly show that the reporter is recruited during expansion phases but lost in stalls (Figure 2A,B). This correlation holds true globally: SCAR reporter intensity along the membrane correlates well with the local membrane expansion speed (Figure 2C) as all pixels with high SCAR reporter are associated with positive instantaneous membrane speed. Note that the small set of pixels with very high membrane speeds and no SCAR (Figure 2C, red arrow) are due to blebs (Zatulovskiy et al., 2014). Figure 2 Download asset Open asset Macropinocytic cups contain a central domain of PIP3 surrounded by a ring of SCAR. (A, B, C) Evidence that fluorescently tagged SCAR complex faithfully marks regions of active actin polymerisation: (A) An aggregation-competent cell moving under an agarose overlay (optimal conditions for visualising pseudopods) showing HSPC300-GFP recruited to the two pseudopods; (B) Kymograph of expansion of the pseudopod arrowed in (A) showing that the SCAR reporter is present during periods of expansion, but absent in the plateaus when the pseudopod is not expanding; (C) Membrane speed and SCAR complex accumulation are positively correlated. The HSPC300-GFP signal and local membrane speed was measured at 100 points along the membrane of a motile cell. of independent cells was and as a arrow data points with high levels of SCAR and positive arrow data points due to which are and expand than pseudopods (Zatulovskiy et al., 2014). (D) SCAR is recruited as a ring to the of macropinocytic cups. The show top and side of a surface of a cell with three macropinocytic cups and the the cell with a SCAR (E) SCAR is recruited to pseudopods in distinct not as a ring. of the 3D surface is a by small that the cell membrane. (F) SCAR is recruited to the of an intense PIP3 in the macropinocytic cup. The white in the panel to the of the in the right (G) 3D of the cell in the previous (H) F-actin is in the macropinocytic cup and does not the of SCAR. Ax2 cells were used in all HSPC300 was used as a marker for the SCAR complex, as a reporter for and as a reporter for 3D were from taken on a In in the reporter a ring of active SCAR around the of macropinocytic cups (Figure and Video The presence of a ring could not be by imaging the actin cytoskeleton as actin are rather the cup (Figure These circular SCAR structures are not in pseudopods, where 3D show the of SCAR as in (Figure Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg The rim of macropinocytic cups is by a of SCAR. is a vegetative Ax2 cell expressing the SCAR marker A was using a and using a point spread panel a surface of the cell and the right panel a 3D of the signal using maximum intensity The of these the question of how SCAR molecules are to the ring We PIP3 using a reporter that the of to (Insall et al., This revealed a of SCAR they follow the of or et al., of In all macropinocytic cups of the cup a of PIP3 and SCAR is present as a ring around this to its centre (Figure and G and Video Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Macropinocytic cups are by a of PIP3 that is by a of SCAR. is a vegetative Ax2 cell expressing the SCAR marker HSPC300-GFP and the PIP3 marker A was using a and using a point spread Image is a 3D of the signal using maximum intensity This was in a larger sample by intensity at the centre and rim of macropinocytic cups from cells in 3D (Figure cup high levels of PIP3, but SCAR followed the rim of the cup with the of the SCAR reporter than cytosolic background while signal at the centre of the PIP3 was not different from the cytosolic This is also in but is as the SCAR ring appears as in the from microscopy. Figure 3 Download asset Open asset SCAR is present at the but not the centre of the macropinocytic cup. (A, of PIP3 and SCAR in macropinocytic cups identified (A) of Macropinocytic cups were identified from 3D and the of their arrow) and was intensity along the cell membrane of the macropinocytic cup was and the intensity was measured at the and (B) PIP3 and SCAR intensity at and of identified macropinocytic cups. the the and indicate the of the of SCAR in PIP3 (C) of PIP3 intensity of the markers was measured for cells. PIP3 patches were as membrane regions where the intensity is than one and these regions are in (D) of intensity at the and of identified PIP3 the the and indicate the of the (E) SCAR and PIP3 reporter In this the intensity of in all membrane pixels was of the morphological structure in which they The the data from cells. Vegetative Ax2 cells expressing the SCAR complex reporter HSPC300-GFP and the reporter were used in all The marks significant differences We further tested the spatial PIP3 and SCAR in two not of macropinocytic cups. In the a number of growing axenic cells was as Membrane with intensity of the PIP3 reporter than cytosolic background one were as PIP3 patches, and the associated SCAR signal was In all cases SCAR is and at and at their (Figure In the the intensity of the SCAR and PIP3 was from the of a number of growing cells and the results as a (Figure In pixels with high PIP3 the SCAR signal is and in pixels with high SCAR, PIP3 is This method cannot show whether high SCAR and PIP3 pixels are but it does confirm that SCAR and PIP3 do not but instead are SCAR is associated to the of PIP3 patches macropinocytic cup The of a de novo macropinocytic cup is in Figure (Video The PIP3 first at t = 1 and this is by of SCAR. As the of PIP3 grows the SCAR associated with its right up to of the macropinocytic cup, which the SCAR signal quickly and the is The SCAR is not at the of the patches Figure 4 Download asset Open asset A SCAR ring PIP3 patches their (A) Detail of a cell initiating a de novo macropinocytic cup. of SCAR the PIP3 at all is in seconds. (B) Kymograph of the intensity along the membrane of a vegetative cell several macropinocytosis SCAR is associated with the of PIP3 patches during their entire and with their (C) of SCAR signal during macropinocytosis. A was through the of the macropinocytic cup and into the for of a time of the of a macropinocytosis were resulting in as in panel the Numbers indicate independent macropinocytosis (E) The SCAR signal at the of macropinocytic cups is present from to The intensity at the macropinocytic cup was and