A role for SNX5 in the regulation of macropinocytosis
- Jet Phey Lim†1,
- Jack TH Wang2Email author,
- Markus C Kerr2,
- Rohan D Teasdale2 and
- Paul A Gleeson1Email author
© Lim et al; licensee BioMed Central Ltd. 2008
Received: 03 June 2008
Accepted: 14 October 2008
Published: 14 October 2008
The mechanisms and components that regulate macropinocytosis are poorly understood. Here we have investigated the role of sorting nexin 5 (SNX5) in the regulation of macropinocytic activity.
SNX5 is abundantly expressed in macrophages, cells very active in macropinocytosis, and is recruited onto newly-formed macropinosomes. LPS treatment of bone marrow-derived macrophages resulted in a 2.5 fold decrease in macropinosome formation that correlates with a reduction in the levels of SNX5. To investigate the relationship between SNX5 levels and macropinocytic activity we examined the formation of macropinosomes in HEK-FlpIn cells stably expressing GFP-SNX5. Constitutive macropinocytosis was increased ~2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Furthermore, EGF stimulation resulted in a significant increase in macropinocytosis and there was also a 2.0 fold increase in the generation of macropinosomes in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. SNX5, which interacts specifically with PtdIns(3)P and PtdIns(3,4)P2 through its PX domain, was recruited to regions on the plasma membrane containing EGF receptor or positive for PtdIns(3,4)P2 as detected with the PH domain of TAPP1. Treatment with AG1478, an EGF receptor specific tyrosine kinase inhibitor, prevented the recruitment of SNX5 to the cytosolic face of the plasma membrane and inhibited the formation of macropinosomes in response to EGF treatment.
Based on these data, we propose that SNX5 requires the generation of phosphoinositides for recruitment to the plasma membrane and, moreover, influences the level of macropinocytic activity.
Macropinocytosis is an endocytic process that enables cells to internalize large amounts of solutes from the external environment. Macropinosomes are generated from the base of actin-mediated membrane ruffling when the lamellipodia folds back onto itself thereby forming very large endocytic structures. Macropinosomes are heterogeneous in size and generally considered to be > 0.2 μm in diameter [1, 2], a size considerably larger than clathrin-coated vesicles. The formation of macropinosomes is largely a signal dependent process that is transiently induced by growth factors such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) or tumour promoting factors such as phorbol myristate acetate (PMA) [3–6]. Given the large size of macropinosomes, this unique organelle provides an efficient route for non-selective entry of solute macromolecules as well as considerable amounts of plasma membrane into the cell .
Macropinocytosis is important in a range of physiological processes. For example, macropinocytosis has a role in the down-regulation of signalling from the plasma membrane  and, because of its dependence upon membrane ruffling, in cell motility . Consequently macropinocytosis is very relevant to tumour progression and metastasis. In addition, this endocytic pathway is the primary mechanism by which macrophages and dendritic cells sample their immediate environment for circulating antigens . Indeed, the major antigen presenting cells, namely macrophages and dendritic cells, are highly active in macropinocytosis . For example, macrophages undergo extensive constitutive macropinocytosis, internalizing up to 200% of their surface area every hour , patrolling and sampling the environment for their role as antigen presenting cells of the immune system. Also immature dendritic cells are able to macropinocytose large quantities of exogenous solute as part of their sentinel function . On the other hand, maturation of dendritic cells is associated with down-regulation of macropinocytosis to maximise the presentation of captured antigen . In addition to antigen uptake, macropinocytosis is also considered important in the chemotactic response of neutrophils and macrophages . This endocytic pathway is also utilised by various pathogens such as Salmonella and Shigella to gain entry into host cells .
Despite the physiological relevance of macropinocytosis, the molecular basis for the regulated formation and maturation of macropinosomes is very poorly understood. Macropinosome formation in a range of cell types has been shown to be phosphoinositide-3 kinase dependent  and unlike the relatively well-characterised phagosome, its regulation is receptor-mediated. As endocytic compartments mature, the bulk of their protein constituents are maintained . Peripheral membrane proteins are differentially recruited in a temporally dependent manner in response to a shift in the organelle's phosphoinositide composition. The phosphoinositides (PtdIns) have become the focus of intense interest as they are linked to a range of cell signalling events and are key regulators of intracellular membrane trafficking. Whilst PtdIns(3,4)P2 , PtdIns(4,5)P2  and PtdIns(3,4,5)P3  are traditionally considered to be associated with signalling at the plasma membrane in response to extracellular stimuli, the monophosphorylated phosphoinositide PtdIns(3)P, is implicated in the membrane trafficking of the endosomal system.
Sorting nexins are a large family of proteins characterised by the presence of a phox (PX) domain at the amino terminus. The modestly conserved PX domain is a sequence of 70 to 120 residues that has been shown to bind to various phosphoinositides hence the PX domain confers phosphoinositide specificity to the protein . Sorting nexins have roles in endocytic trafficking events [19–21]. One such sorting nexin is SNX5, a 404 residue protein that contains a central PX domain and large C-terminal domain predicted to include a BAR domain [22, 23], a domain believed to be involved in membrane curvature and tubulation. SNX5 was first discovered based on its homology to SNX1 and subsequently shown to associate with early endosomes. We have previously reported that the PX domain of SNX5 binds specifically to PtdIns(3)P and PtdIns(3,4)P2  and SNX5 is associated with newly-formed macropinosomes following EGF stimulation . After EGF stimulation of HEK293 cells, large EEA1- and SNX5-positive structures were detected in close proximity to membrane ruffles which labelled with fluid phase markers , characteristics that define macropinosomes. We have also exploited the association of SNX5 with macropinosomes to track their maturation in live cells . Our previous work showed that the association of SNX5 with macropinosomes is highly dynamic and SNX5 is localised not only to the macropinosome body but also tubules that extend and depart from the newly-formed macropinosomes . Here, we present data showing that SNX5 modulates macropinocytic activity as well as evidence that the generation of phosphoinositides is involved in the recruitment of SNX5 to the plasma membrane prior to macropinosome formation.
Antibodies, conjugates and DNA constructs
Rabbit anti-mouse SNX5 was raised against a synthetic peptide corresponding to the N-terminal 35 residues of mouse SNX5, with an additional Cys on the C-terminus, and affinity purified on a column of peptide conjugated to SulfoLink Coupling Gel (Pierce, USA). Monoclonal mouse antibodies to human EEA1 were from BD Transduction Laboratories (California, USA). Mouse monoclonal anti-human EGF receptor (EGF-R) antibody (clone 528) has been described . Monoclonal mouse antibodies to α-tubulin, goat anti-rabbit IgG Alexa Fluor® 488, goat anti-mouse IgG Alexa Fluor® 568, goat anti-mouse IgG Alexa Fluor® 647, biotinylated EGF complexed to Alexa Fluor® 555 streptavidin and Texas-Red-X Phalloidin were purchased from Molecular Probes (Oregon, USA). Horse-radish peroxidase conjugated rabbit anti-mouse and swine anti-rabbit immunoglobulins were from Dako (Glastrup, Denmark). Full length human EGF-R was cloned into the XbaI sites of pCI-neo. The PH domain of TAPP1 (aa 95–400) was from Dr. S. Dowler (MRC, University of Dundee, Dundee, UK).
Cell culture and transfection
HEK-FlpIn (Invitrogen, USA) and HEK-GFP-SNX5 stable cells were generated, cultured and transfected as previously described . Primary bone marrow-derived macrophages (BMMs) were generated by culture of bone marrow cells for 7 days in M-CSF as described . BMMs were activated with 10 ng/ml LPS (Sigma-Aldrich, Australia) up to 24 h before fixation in 4% paraformaldehyde (PFA) or preparation for immunoblotting.
Cell monolayers were cultured for various time periods in the presence of 100 μg/ml dextran (10,000 MW) conjugated to tetramethylrhodamine (TR-dextran) (Molecular Probes, USA). The cells were then washed with media at 4°C and fixed in 4% PFA. Images were captured using an LSM 510 Meta confocal microscope and quantified for macropinosomes using an automated image analysis pipeline in ImageJ 1.37v (NIH). The 'Substract Background' functionality was executed, and images thresholded to a binary image. Dextran-positive structures at least 0.5 μm in diameter were quantified through the 'Analyze Particles' feature.
For analysis of macropinosomes using endosomal markers, cell monolayers were washed twice using PBS, serum-starved for 16–24 h at 37°C and stimulated with EGF (100 ng/ml) (Invitrogen, USA) at 37°C for 10 min. Cells were then washed twice in PBS, fixed in 4% PFA and stained using mouse anti-human EEA1 followed by goat anti-mouse Alexa Fluor 568. Macropinosomes were identified based on their size (>1 μm) , circular structure and the staining pattern for EEA1 which resembled a halo. The mean number of macropinosomes from a triplicate of 100 cells was determined. Each experiment was repeated at least twice without knowledge of the identity of samples. Counts were performed using the Zeiss Axioplan 2 microscope.
EGF Internalization assay
HEK-GFP-SNX5 cells, transiently transfected with pCI-neo-EGF-R for 24 h, were serum-starved for 16–24 h at 37°C. Cells were washed twice using ice-cold PBS and incubated with 100 ng/ml Alexa Fluor 555 conjugated-EGF (Molecular Probes, USA) in serum-free media for 10 min on ice. Excess EGF was removed by washing the cells three times in ice-cold PBS. Cells were resuspended in ice-cold serum-free media (in the absence and presence of drugs) and the cells were then incubated at 37°C for the indicated duration to allow EGF internalization. Cells were then fixed and processed for immunofluorescence analysis.
Cell monolayers were washed twice with PBS and serum-starved for 16–24 h at 37°C. Cells were treated with 100 nM AG1478 (Calbiochem, USA) (from a 10 mM stock in DMSO) for 15 min at 37°C. EGF (100 ng/ml) (Invitrogen, USA) was then added to the cells in the presence or absence of AG1478 at 37°C for the indicated durations. Cells were then washed twice with PBS and processed for immunofluorescence.
Cell monolayers grown on poly-L-lysine coated coverslips were washed twice in PBS and fixed in 4% PFA for 15 min. Cells were processed for immunofluorescence as previously described  and examined using either a Zeiss Axioplan 2 microscope or a Leica TCS SP2 laser unit and Leica Confocal Software version 2.61 or a Zeiss LSM 510 Meta confocal microscope. For multi-colour labelling, images were collected independently.
Endosome-lysosome fusion assay
Late endosomes and lysosomes of cells were labelled with fluorescently-conjugated dextran as described previously . Briefly, cells grown on poly-L-lysine coated coverslips were incubated with 100–200 μg/ml dextran conjugated to Alexa 647 (MW 10,000 Da) (Molecular Probes, USA) for 4 h at 37°C. The cells were then thoroughly washed and further incubated in normal growth media for 16 h to allow the dextran to accumulate in the late organelles of the endosomal system. The cells were then pulsed with 100–200 μg/ml dextran conjugated to TR (MW 10,000 Da) (Molecular Probes, USA) for 5 min at 37°C to label macropinosomes, washed and chased in normal media for 4, 10, 15 or 20 min before being fixed in 4% PFA. The cells were imaged on an LSM 510 Meta confocal microscope. The proportion of TR-labelled macropinosomes that had acquired content from the late endosomes/lysosomes (ie. Alexa 647-positive) were scored for >50 cells per time point.
Proteins were resolved on 10% SDS-polyacrylamide gels and transferred onto Immobilon-P PVDF membranes (Millipore, USA) according to the manufacturer's instructions and immunoblotting performed as described . SNX5 was visualized with rabbit anti-SNX5 followed by horseradish peroxidase-conjugated anti-rabbit secondary antibody. Bound antibodies were detected using chemiluminescence and images were captured using Gel-Pro™ Analyser version 4.5 software. Bound antibodies were stripped from the membrane using PVDF membrane stripping buffer (25 mM glycine, 1% (w/v) SDS, PBS, pH 2) as necessary. Membrane was then washed and blocked, as described above, prior to subsequent incubation with different primary antibodies.
Data were analysed by an unpaired Student's t-test, two-tailed.
SNX5 is located on macropinosomes in both cultured and primary cells
SNX5 modulates macropinocytosis
To directly explore the role of SNX5 in macropinocytosis we have used HEK-FlpIn cells which stably express SNX5-GFP. This cell line was the previously described HEK-FlpIn cell line transfected with pcDNA5/FRT-EGFPSNX5, referred to as HEK-GFP-SNX5 . HEK-GFP-SNX5 is a polyclonal cell line where the construct is integrated in a defined chromosomal location and therefore avoids differences due to clonal variation. HEK-GFP-SNX5 has overall ~2 fold-increase in the level of SNX5 compared with HEK-FlpIn cells , an increase in SNX5 level which was consistent throughout the course of these studies, as monitored by immunoblotting.
We then determined the impact of the elevated level of SNX5 on macropinocytosis after EGF stimulation. After a 5 minute EGF stimulation of serum-starved cells, HEK-GFP-SNX5 cells exhibited a 2.0 fold increase in the number of macropinosomes compared to HEK-FlpIn cells (p < 0.005), utilizing the Dextran-uptake assay (Fig 2A). These data demonstrate that cells expressing elevated levels of SNX5 have an increase in the capacity for macropinocytosis. Moreover, the increase in both constitutive and receptor stimulated-macropinocytosis as a consequence of increased levels of SNX5 indicates that this molecule plays a role in the generation of these endocytic structures.
Next we investigated the relationship between EGF-R signalling and macropinocytosis in the stable HEK-GFP-SNX5 cells using AG1478, an EGF-R specific tyrosine kinase inhibitor that prevents EGF-R activation . Significantly, treatment of cells with AG1478 inhibited the increase in the number of macropinosomes normally observed when cells are treated with EGF alone (Fig. 2B). DMSO, the carrier for AG1478, had no impact on the level of macropinosomes at the same concentration (Fig 2B). Similar results were obtained in three separate experiments. The data demonstrate that the increased macropinocytic activity of HEK-GFP-SNX5 cells following EGF binding is in response to cell signalling events.
SNX5 functions in the formation of macropinosomes
Activation of EGF-R results in the production of PtdIns(3,4,5)P3 and its breakdown product, PtdIns(3,4)P2. We have shown that the PX domain of SNX5 binds to both PtdIns(3)P as well as PtdIns(3,4)P2. To determine the relationship of PtdIns(3,4)P2 generated from activated EGF-R and the plasma membrane recruitment of SNX5, we have used the PtdIns(3,4)P2-specific probe, mCherry-TAPP1-PH domain , to determine the location of PtdIns(3,4)P2 at the plasma membrane. HEK-GFP-SNX5 cells were transfected with mCherry-TAPP1-PH domain, then the cells serum-starved and treated with EGF for 5 min. mCherry-TAPP1-PH was located on the plasma membrane of EGF-stimulated cells, but not non-stimulated cells, and showed co-localization with GFP-SNX5, demonstrating that SNX5 was recruited to regions of the plasma membrane rich in PtdIns(3,4)P2 after EGF stimulation (Fig. 5B). Significantly, treatment of EGF-stimulated cells with AG1478 resulted in the inhibition of SNX5 recruitment to the plasma membrane (Fig. 5C). Qualitative cell analysis showed a reduction from ~57% to 20% of cells with detectable SNX5 at the cell surface (Fig. 5C). The residual 20% positive cells represents a level similar to that observed in non-stimulated cells. Therefore, inhibition of EGF-R tyrosine kinase activity by AG1478 inhibits the recruitment of SNX5 to the plasma membrane and the generation of macropinosomes. Taken together we propose that activation of EGF-R and its effector kinase PI3K are required for SNX5 recruitment to the plasma membrane and subsequent generation of macropinosomes that internalize receptor-EGF complexes.
The mechanisms that drive macropinocytosis are poorly defined. Our previous studies showed that SNX5 associates with newly-formed macropinosomes following EGF stimulation . Given the potential of sorting nexins to have membrane binding and bending properties, mediated by the PX and BAR domains, SNX5 represented a potential regulator of this endocytic process. SNX5 is recruited to the plasma membrane following receptor stimulation and is associated with newly-formed macropinosomes derived from membrane ruffles. In this study we have analysed the impact of SNX5 on numerous aspects of the macropinocytic process. Our results demonstrate that SNX5 is present in abundant levels in primary macrophages that possess high intrinsic macropinocytic activity and that on reduction in macropinocytosis, cell levels of SNX5 are reduced also. Moreover, stable overexpression in HEK293 cells resulted in the recruitment of SNX5 to the cell surface following EGF stimulation and in a significant increase in the number of newly-formed macropinosomes. Upon abrogation of the cell-surface recruitment of SNX5 via inhibition of EGF-R tyrosine kinase activity, the generation of macropinosomes is substantially reduced. Collectively the data indicates SNX5 is a molecular component of macropinocytosis specifically involved in the biogenesis and formation of macropinosomes.
The PX domain of SNX5 has the capacity to bind both PtdIns(3)P and PtdIns(3,4)P2. . The PI binding specificity of SNX5 contrasts with SNX1 which binds selectively PtdIns(3)P and PtdIns(3,5)P2, also based on liposome binding assays . The plasma membrane recruitment of SNX5 following EGF stimulation is likely to be dependent on an interaction with PtdIns(3,4)P2. Cell signalling is essential for the recruitment of SNX5 to the cytosolic face of the plasma membrane, as AG1478 inhibited the movement of SNX5 to the plasma membrane, presumably due to the lack of PtdIns(3,4,5)P3 and its breakdown product PtdIns(3,4)P2.. However, AG1478 does not prevent EGF-R internalization via the clathrin-dependent pathway (unpublished observations), indicating, at least in this system that receptor dimerization is sufficient for clathrin-mediated endocytosis, and consistent with previous findings .
Given the association of EGF signalling and the behaviour of SNX5, it is formally possible that SNX5 is modulating the EGF-R signalling cascade and thereby influencing macropinocytosis indirectly. However, our data argues for a direct role of SNX5 in enhancing the early events of macropinocytosis rather than potentiating EGF-R signalling. Firstly, constitutive macropinocytosis in the absence of EGF stimulation is increased >2 fold in HEK-GFP-SNX5 cells compared with parental HEK-FlpIn cells. Secondly, the kinetics of macropinosome maturation and the rate of EGF-R degradation is similar in parental and HEK-GFP-SNX5 cells following EGF stimulation, indicating SNX5 is promoting the early events driving the biogenesis of macropinosomes, but does not influence the rate of maturation and delivery to lysosomes.
A number of the SNXs can form homodimers and heterodimers in vitro and in vivo. As SNX5 has a coiled-coil C-terminal and a predicted BAR domain, it is likely that it exists as a dimer. We have previously demonstrated that SNX5 does not form a homodimer but can associate with SNX1 and that association of SNX5 with endosomes appears to be dependent on the formation of SNX5/SNX1 heterodimers . SNX5 is selectively recruited to membrane ruffles of activated cells and is not associated with SNX1 . Therefore it is likely that SNX5 exists in two pools, one pool independent of SNX1 and another pool as a heterodimer with SNX1. We propose that a SNX1-independent pool of SNX5 is recruited to the plasma membrane to initiate the formation of macropinosomes whereas the SNX5/SNX1 pool is recruited to a subdomain of maturing macropinosomes to promote tubulation. Furthermore, the differential recruitment of SNX5 to either plasma membrane or nascent macropinosomes is likely to be dependent on the specificity of phosphoinositide binding. Clearly further studies are required to identify the partners of SNX5.
Based on our published data [24, 25] and the data above, our model for macropinosome maturation is as follows. Following receptor stimulation, GFP-SNX5 is recruited to discrete patches on the cytoplasmic surface of the plasma membrane via its phosphoinositide binding PX domain. There is a direct relationship between the level of cell surface SNX5 protein and macropinocytic activity, highlighting the role for this sorting nexin in driving this signal-activated endocytosis pathway. Newly-formed SNX5-positive macropinosomes recruit EEA1 and the retromer complex, possibly mediated by PtdIns(3)P arising from the hydrolysis of PtdIns(3,4)P2. The BAR domain of SNX5, in a complex with other sorting nexins, has the potential to distort the limiting membrane of the macropinosome into extensive tubular elements that subsequently separate from the macropinosome and traffic in a microtubule-dependent manner to perinuclear endosomes. As the tubular structures depart from the macropinosome there is a reduction in the surface area of the macropinosome, which matures into a Rab7-positive structure and eventually fuses with late endosomes/lysosomes. The identification of machinery that regulates this process now provides the ability to directly assess the biological role of macropinocytosis in a range of physiological settings.
macrophage colony-stimulating factor
epidermal growth factor
sorting nexin 5
dextran conjugated to tetramethylrhodamine
green fluorescent protein
bone marrow-derived macrophages
HEK293 cells stably expressing SNX5-GFP
This work was supported by funding from the National Health and Medical Research Council of Australia. JPL is supported by an Endeavour International Postgraduate Research Scholarship and Melbourne International Research Scholarship. JTHW is supported by an Australian Postgraduate Award and RDT is supported by an NHMRC Senior Research Fellowship. The authors would like to thank Mr Allan Burrows for generously supplying the bone-marrow derived macrophages used in this study and Miss Casey Barnes for assistance quantifying macropinosome-LE/Lysosome fusion.
Confocal microscopy was performed at the Australian Cancer Research Foundation (ACRF)/Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology, which was established with the support of the ACRF.
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