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Vol. 19, Issue 4, 1717-1726, April 2008
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Program in Cell Biology, Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5G1X8
Submitted July 9, 2007;
Revised December 27, 2007;
Accepted January 30, 2008
Monitoring Editor: Erika Holzbaur
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R-mediated phagocytosis. We show that several septins are expressed in RAW264.7 and J774 mouse macrophage cell lines and that SEPT2 and SEPT11 are colocalized with submembranous actin-rich structures during the early stages of FcR-mediated phagocytosis. In addition, SEPT2 accumulation is seen in primary human neutrophils and in nonprofessional phagocytes. The time course of septin accumulation mirrors actin accumulation and is inhibited by latrunculin and genistein, but not other inhibitors of phagocytosis. Inhibition of septin function by transient expression of the BD3 domain of BORG3, known to cause septin aggregation, or depletion of SEPT2 or SEPT11 by RNAi, significantly inhibited FcR-mediated phagocytosis of IgG-coated latex beads. Interestingly, this occurred without affecting the accumulation of actin or the actin-associated protein coronin-1. These observations show that, although not necessary for actin recruitment, septins are required for efficient FcR-mediated phagocytosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R) do so via a highly conserved extracellular Fc-binding domain (Ravetch and Kinet, 1991
). A number of key events take place between the time of initial binding of IgG-coated particles to the cell surface and the eventual degradation of these pathogens within intracellular phagosomes. The first notable event is the clustering and subsequent tyrosine phosphorylation of FcRs. Although the receptors themselves do not contain any intrinsic tyrosine kinase activity, several protein tyrosine kinases are known to be activated upon the clustering of FcRs (Greenberg et al., 1994; Indik et al., 1995b). Phosphorylation of tyrosine residues within the conserved immunoreceptor tyrosine activation motif (ITAM) provides important docking sites for SH2-containing molecules, most notably the tyrosine kinase Syk (Greenberg et al., 1994, 1996; Indik et al., 1995a; Crowley et al., 1997; Kiefer et al., 1998). Ultimately these early signaling events lead to local remodeling of the submembranous actin cytoskeleton (Greenberg et al., 1990), whose intracellular force-generating capabilities contribute, along with the targeted delivery of new membrane (Bajno et al., 2000; Niedergang et al., 2003), to the growth of the plasma membrane around the particle. Interestingly, expression of the FcRIIA receptor in nonphagocytic cells, such as COS or Chinese hamster ovary (CHO) cells, is sufficient to confer on them phagocytic properties (Indik et al., 1995a).
Although FcR-mediated phagocytosis is an actin-dependent process, the role of other cytoskeletal elements is not particularly well understood. Several members of the septin family have been shown to be colocalized with filamentous actin (Kinoshita et al., 1997, 2002; Xie et al., 1999), possibly by their binding to myosin (Joo et al., 2007). Septins are a highly conserved family of filamentous GTPases that were originally identified in Saccharomyces cerevisiae as factors controlling bud-site selection (for reviews see Gladfelter et al., 2001; Longtine and Bi, 2003). However, since their discovery as factors that control cytokinesis in animal cells (Neufeld and Rubin, 1994; Kinoshita et al., 1997; Surka et al., 2002), numerous observations suggest that septins may also regulate other important cellular functions including cell membrane dynamics and vesicle fusion events (Fares et al., 1995, 1996; DeMarini et al., 1997; Hsu et al., 1998; Beites et al., 1999; Joo et al., 2005).
In this study we investigated the role of mammalian septins during FcR-mediated phagocytosis. We find that the murine macrophage cell lines J774 and RAW264.7 express a subset of septin gene products. Antibodies to two of these, SEPT2 and SEPT11, reveal that these proteins transiently accumulate at the phagocytic cup in J774, RAW264.7, and human neutrophils and in CHO or HeLa cells engineered to phagocytose. Inhibition of septin function, either by the use of a dominant inhibitor of septins, or by RNA interference (RNAi)-mediated depletion, significantly inhibits FcR-mediated phagocytosis without affecting the signaling events required for actin accumulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). psiRNA-hH1GFPzeo and psiRNA-hH1gz-Scr (a plasmid containing a control sequence specific for luciferase) vectors were from Invivogen (San Diego, Ca). All restriction enzymes were from New England Biolabs (Beverly, MA). Wortmannin, latrunculin B, and genistein were from EMD Biosciences (San Diego, CA). HeLa cells were obtained from the ATCC (Manassas, VA), and CHO-IIA cells were kindly provided by Dr. Alan Schreiber (University of Pennsylvania, Philadelphia, PA). HP Genome-wide small interfering RNA (siRNA) for human SEPT11 was obtained from Qiagen (Mississauga, ON, Canada) for the following target sequences: hs_SEPT11_2 HP, hereafter called SEPT11 siRNA1, 5'CAAGAGG-AATTGAAGATTAAA3' and hs_SEPT11_5 HP, hereafter called SEPT11 siRNA2, 5'ACGGCTGAAGTTAACCATTGT3'.
DNA Constructs
The construction and utilization of mammalian expression vectors pEGFP-PLC
PH, pRFP-coronin-1, and pEGFP-PM have been described elsewhere (Botelho et al., 2000; Scott et al., 2005; Yan et al., 2005). GFP3-BD3 plasmid (Joberty et al., 2001) was kindly provided by I. G. Macara (University of Virginia, Charlottesville, VA). pmRFP-PLCPH was generated by cutting the PH domain from pEGFP-PLCPH vector using XhoI and BamHI and subcloning the fragment into the corresponding sites of pDsRed-Monomer C1 vector (Clontech, Mountain View, CA). To generate siRNA specific for nucleotides 524–544 relative to the start codon of the human septin 2, the following complementary pair of nucleotides for this region including a hairpin sequence containing a BamHI site were generated to include Acc65I and HindIII at the ends: 5'GTACCTCGAATATTGTGCCTGTCATTGGGATCCCAATGACAGGCACAATATTCTTTTTGGAAA3' and 5'AGCTTTTCCAAAAAGAATATTGTGCCTGTCATTGGGATCCCAATGACAGGCACAATATTCGAG3'. These were cloned into the corresponding sites of psiRNA-hH1GFPzeo vector, expressing short hairpin RNA (shRNA) and green fluorescent protein (GFP) from H1 and CMV promoters, respectively.
Cell Culture and Transfection with DNA or shRNA
Culture conditions for mouse macrophage cell lines RAW 264.7, J774, and CHO cells stably transfected with FcRIIA receptor (CHO-IIA) have been described elsewhere (Hackam et al., 1997; Vieira et al., 2003). CHO-IIA cells and RAW 264.7 cells were transiently transfected by using FuGene 6 (Roche, Nutley, NJ) or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as suggested by the manufacturers. Cells were transfected either with GFP3-BD3 using GFP as a control for 36–48 h or with shRNA-septin 2 using shRNA-control as control for 84–96 h. All other plasmids were transfected for 24 h.
Isolation of Human Neutrophils
Human blood was obtained from healthy volunteers. The neutrophils were purified using Polymorphprep (Accurate Chemical and Scientific, Westbury, NY) gradient separation procedure according to the manufacturer's instructions. Isolated neutrophils were resuspended in PBS containing 1 mM calcium chloride, 1 mM magnesium chloride, and 10 mM glucose at 
1 x 106 cells/ml and used within 6 h of isolation.
Phagocytosis Assays and Inhibitors Treatment
Latex beads were opsonized with 0.8 mg/ml human IgG, incubated for 1 h at 37°C, and washed with phosphate-buffered saline (PBS) 3 times. When noted, the cells were treated with 250 nM wortmannin or 5 µM latrunculin B for 30 min or 100 µg/ml genistein for 3 h before phagocytosis. Opsonized beads were allowed to attach to the cells for 10 min on ice in cold HEPES-buffered RPMI. Unbound beads were washed away with cold PBS. Phagocytosis was started by replacing cold medium with HEPES-buffered RPMI prewarmed to 37°C and incubated at 37°C for the designated time. When necessary, the beads were stained by incubating with Cy5-labeled donkey anti-human IgG on ice for 15 min. Cells were then fixed with 4% paraformaldehyde and permeabilized as described previously (Yan et al., 2005). Endogenous SEPT2 was detected by immunostaining with rabbit anti-human septin 2 primary antibody and Cy3-, FITC-, or Cy5-labeled donkey anti-rabbit secondary antibodies. In the case of SEPT11, cells were fixed for 10 min in 1% paraformaldehyde at 37°C, as above, stained with Cy5 donkey anti-human antibody to detect external beads, and then permeabilized with MeOH at –20°C for 10 min.
For phagocytic index assays, RAW 264.7 or CHO-IIA cells transfected either with GFP-BD3 or with shRNA-septin 2 were treated as above. HeLa cells were transfected with siRNA for 2 d and then retransfected with a plasmid containing the FcIIA-GFP receptor, and phagocytosis was performed on the third day. The phagocytic index was calculated by counting the average numbers of internalized beads per cell and normalized to that of the controls performed the same day. The binding index was calculated by counting the average numbers of bound beads on the cell surface without performing phagocytosis and normalized as per the phagocytic index.
Microscopy
Laser-scanning confocal microscopy was performed using an LSA 510 laser scanning confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY) with 63x or 100x oil immersion objective and analyzed using Photoshop 6.0 (Adobe, San Jose, CA). Spinning disk images were captured on a Zeiss Axiovert 200M equipped with a Quorum confocal spinning disk head (Quorum Technologies, Guelph, ON, Canada).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IIA receptor. As can be seen in Figure 1, SEPT2, SEPT6, SEPT10, and SEPT11 were abundantly expressed in all four cell types. SEPT9 was detectable in CHO cells but much less so in brain and not at all in RAW 264.7 or J774 cells. SEPT7 and was not studied here because of the lack of a suitable antibody, and SEPT12 is not shown because it is only abundantly expressed in testes and was not present in the brain or any of the cell lines (Steels et al., 2007). Because septins typically associate in coimmunoprecipitating complexes (for example, see Kinoshita et al., 2002) and because our antibodies to SEPT2 and SEPT11 are superior to anti-SEPT6 and SEPT10 for immunocytochemistry, we chose to focus on SEPT2 and SEPT11 for the remaining studies.
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-Receptor–mediated Phagocytosis: SEPT2 and SEPT11 associate with forming phagosomes; Kinoshita et al., 1997; Xie et al., 1999; Surka et al., 2002). To determine whether septins may play a role in FcR-mediated phagocytosis, another actin-dependent cellular process, we first examined their distribution in cells phagocytosing latex beads via FcRIIA receptors (Figure 2). Robust accumulation of SEPT2 was observed in the murine macrophage cell lines J774 and RAW 264.7, although a stronger accumulation was observed in the former. In J774 cells SEPT2 accumulated all around the particle (Figure 2, a and b), whereas in RAW264.7 cells the accumulation was most typically seen at the base of the phagosome pedestal (Figure 2, c and d). Strong accumulation was also observed in primary human neutrophils (Figure 2, e and f) and in CHO-IIA cells stably expressing the FcIIA receptor (Figure 2, g and h). It should be noted that the time course of phagocytosis in each of these models is quite different, with neutrophils being the fastest and CHO-IIA being the slowest. To determine if this was a specific accumulation of SEPT2, or involved multiple septins, likely in a complex, we also stained for SEPT11 (Figure 2, i and j). Although immunocytochemistry with this antibody is inferior to that of SEPT2, a clear accumulation of SEPT11 could be seen associated with the nascent phagosome in CHO-IIA cells.
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R-mediated phagocytosis, we next explored the time course of their appearance on the phagosome. Given the slower kinetics of CHO-IIA phagocytosis, these cells were chosen to monitor the internalization process. CHO-IIA cells were challenged with human IgG-opsonized latex beads for different periods of time at 37°C, fixed, and then stained for filamentous actin using Alexa488-labeled phalloidin and SEPT2. Within 3–5 min, opsonized latex beads are seen engaged in phagocytosis. Unsealed phagosomes were identified by staining for external human IgG after light fixation in the absence of detergent (not shown) and are indicated with open arrows (Figure 3a). The accumulation of filamentous actin in collar-like structures is characteristic of nascent phagosomes (Kolotila and Diamond, 1988; Greenberg et al., 1990). Fully internalized particles not detected by immunostaining for human IgG in nonpermeabilized cells (filled arrows, Figure 3a) appeared to concurrently lose both actin and SEPT2.
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), and these lipids are also known to influence actin polymerization. We therefore examined the timing of PIP2 accumulation at the phagosome relative to that of SEPT2. In this case CHO-IIA cells were transiently transfected with pEGFP-PLCPH (Scott et al., 2005) and examined for 10 min after initiation of phagocytosis. As seen in Figure 3b, SEPT2 accumulates within 3 min to unsealed nascent phagosomes (open arrows) in concert with PIP2 accumulations detected by pEGFP-PLCPH. As with actin, the SEPT2 and PIP2 levels decrease on sealed phagosomes with a similar time course (solid arrows, 10 min).
SEPT2 Accumulation Depends on Actin
To begin to examine the signals that are responsible for septin accumulation at the nascent phagosome, CHO-IIA cells were treated with inhibitors capable of blocking phagocytosis. Wortmannin, a potent inhibitor of type I and III PI3 kinases, partially inhibits phagocytosis of large particles (Araki et al., 1996; Cox et al., 1999). After confirming that wortmannin had inhibited phagocytosis under the conditions used (not shown), there remains robust accumulation of actin at the base of bound opsonized beads, and SEPT2 can be seen to accumulate at these sites (Figure 4, a and b), indicating that PI3-kinase activity is not required for SEPT2 accumulation. We therefore examined an upstream event in the signaling process, the activation of src-family tyrosine kinases (Greenberg et al., 1993). Using the general inhibitor of tyrosine kinases, genistein, we found that in the majority of the cells actin accumulation at the phagosome was inhibited. In these cells the accumulation of SEPT2 was also significantly reduced (Figure 4, c and d), consistent with the role of tyrosine kinases in early stages of actin remodeling.
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SEPT2 Function Is Required for Efficient Phagocytosis
The transient accumulation of SEPT2 at the phagosome raised the possibility that it may provide a necessary function for phagocytosis. To determine if septins are required for phagocytosis, we used two approaches to inhibit their function. First, we took advantage of a dominant inhibitor known to inhibit septins by causing their aggregation. Borg proteins are Cdc42 effectors, and one of the Borg isoforms, Borg3, was shown to bind to septins via its third Borg homology domain, BD3. Overexpression of BD3 fused to GFP was shown to result in the coaggregation of GFP-BD3 and septins into cytoplasmic structures in the majority of cells (Joberty et al., 2001). The aggregation depletes the available septins, thereby inhibiting their function. We therefore overexpressed GFP-BD3 in CHO-IIA cells and found cytoplasmic aggregates in which SEPT2 and GFP colocalized in 50% of transfected cells (data not shown), similar to that previously reported (Joberty et al., 2001), but for all of these studies we limited our analysis to those cells exhibiting a clear cytoplasmic aggregation of GFP. Transfected cells were exposed to opsonized latex beads, allowed to phagocytose at 37°C for 30 min, and unbound beads were washed away. After fixation without permeabilization, the cells were stained with anti-human IgG antibodies to detect external particles. As can be seen in Figure 5a, cells overexpressing GFP-BD3 (arrowhead) were unable to phagocytose particles, whereas those transfected with GFP alone (Figure 5b, arrowhead) were fully able to internalize numerous beads. To ensure that this was not an artifact of the CHO-IIA model system, we repeated the same experiments in RAW 264.7 cells. As shown in Figure 5c, transfected RAW 264.7 cells (arrowhead) were unable to ingest bound particles (open arrow), whereas neighboring untransfected cells phagocytosed particles completely (filled arrow). Again, control GFP-transfected cells, like their untransfected neighbors, were fully capable of robust phagocytosis (Figure 5d).
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). In cultures transfected with the SEPT2 shRNA vector containing a GFP reporter 70% of the cells were effectively transfected, and by Western blotting we see an 50% decrease in SEPT2 levels (Figure 6d). In SEPT2-shRNA–transfected cells (in Figure 5e, GFP-positive cells, indicated with a white dotted line) there were few internalized beads and many more that could be detected by antibodies in nonpermeabilized cells (open arrow). In contrast, neighboring GFP-negative cells were capable of ingesting particles efficiently (filled arrows). Transfection of the vector containing a scrambled sequence had no effect on phagocytosis efficiency (Figure 5f). Similar results were obtained when RAW 264.7 cells were used, although the low efficiency of transfection precluded their quantification.
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IIA receptor along with GFP-BD3 into CHOIIA cells and found that, like SEPT2, SEPT11 accumulates in the BD3 aggregate and phagocytosis is inhibited in these cells (Figure 5g). This inhibition of phagocytosis could be due solely to the depletion of SEPT2, which is also found in the aggregate, so to determine if SEPT11 is also required for phagocytosis we set out to deplete it by siRNA. Unfortunately, the sequence of SEPT11 in Chinese hamster is not known, and available rodent SEPT11 siRNA sequences did not appear to work in this cell line (data not shown). However, HeLa cells, like CHO cells, become phagocytic when forced to express the FcIIA receptor. We therefore tested two commercial siRNA reagents specific for distinct target sequences and found that they were efficient at depletion of human SEPT11 protein from HeLa cells (Figure 6f). We therefore transfected HeLa cells with siRNA specific for human SEPT11, subsequently transiently transfected a GFP-tagged version of the FcIIA receptor, and measured phagocytosis in the GFP-positive cells. As can be seen in Figure 5h, FcIIA receptor-GFP-positive cells, which were depleted of SEPT11, failed to internalize beads, in contrast to the negative control siRNA. The same results were obtained with both siRNA sequences (not shown). The results of three independent experiments were quantified and are shown in Figure 6. As can be seen in Figure 6, a–c and e, overexpression of Borg3 GFP-BD3 or shRNA depletion of SEPT2 and siRNA depletion of SEPT11 resulted in a significant decrease of between 50 and 70% of the phagocytosis without affecting the ability of the particles to bind to the phagocytic cells, for both the engineered phagocytic CHO-IIA and HeLa cells and the macrophage cell line RAW 264.7.
SEPT2 Depletion Does Not Affect Actin Accumulation
Septins and actin are closely associated in cells (Kinoshita et al., 1997; Xie et al., 1999) and functional interdependence has been suggested to exist between them. Specifically, depletion of septins results in loss of stress fibers, suggesting that they stabilize actin filaments, and actin filaments appear to serve as templates for septin filament assembly (Kinoshita et al., 2002; Schmidt and Nichols, 2004). We therefore examined the effects of septin inhibition on actin accumulation at the nascent phagosome. As shown in Figure 7, a–c, in CHO-IIA cells expressing GFP-BD3 (Figure 7a), when particles bind to these cells there is robust accumulation of actin (arrows, Figure 7c) despite the failure to detect SEPT2 accumulation (Figure 7b). Similar observations are made with phosphotyrosine accumulation (Figure 7, d–f). This indicates that although septins may influence actin stability to some extent, they are not required for the accumulation of F-actin at the phagocytic cup.
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). We therefore examined the accumulation of coronin-1 by transient transfection of RFP-coronin-1 in CHO-IIA cells expressing GFP-BD3. As seen in Figure 7, g–i, cells exhibiting aggregates of SEPT2 (Figure 7h) still had bound particles to which RFP-coronin-1 was recruited (Figure 7i, arrows). Hence, both the accumulation of F-actin and the recruitment of actin assembly factors appear to occur normally in the absence of functional septins. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or by depletion of SEPT2 or SEPT11 by RNAi, decreased the efficiency of phagocytosis between 50 and 70%. This occurred despite the apparently normal recruitment of actin and actin-associated proteins to the nascent phagosome. Moreover, using a phagocytosis-competent cell line, CHO-IIA and transiently transfected HeLa cells, we were able to show that SEPT2 and SEPT11 are involved in the signaling events initiated by a single class of immunoglobulin receptors, FcRIIA.
The mechanisms recruiting septins to the phagosome are not yet clear, but we and others have previously shown that septins are capable of binding to phosphatidylinositol lipids (Zhang et al., 1999; Casamayor and Snyder, 2003; Rodriguez-Escudero et al., 2005). Septins possess a conserved polybasic region adjacent to the GTPase domain that appears to be responsible for this interaction (Zhang et al., 1999). In light of the importance of PIP2 production (Coppolino et al., 2002; Defacque et al., 2002) and phosphoinositide 3-kinase signaling (Araki et al., 1996) to FcR-mediated phagocytosis, it is possible that one of roles of these lipids is in recruitment of the septins. Alternatively, others have shown that septins can be recruited to actin bundles by adaptor proteins such as anillin (Kinoshita et al., 2002) and myosin (Joo et al., 2007), raising the possibility that the accumulation of septins at the phagosome may be due to their assembly on actin-rich structures. However, anillin resides in the nucleus of interphase cells and would not be anticipated to be available as a linker. Hence, if septins are recruited to the phagosome by the presence of actin bundles, other scaffolding proteins such as myosin may be necessary to provide the linkage.
The exact role of septins in phagocytosis is unclear, but it does not appear to involve the recruitment or assembly of actin filaments in the vicinity of the nascent phagosome. This is somewhat surprising because mutations in yeast septins have been shown to affect the organization of filamentous actin (Adams and Pringle, 1984). In addition, depletion of septins in mammalian cells results in a reduction of actin stress fibers (Kinoshita et al., 2002; Joo et al., 2007), suggesting that septins may play a role in stabilizing these structures. However, we observed significant actin accumulation at the site of FcRIIA receptor engagement, even in the absence of SEPT2 accumulation. In the case of SEPT2 or SEPT11 RNAi treatment, this could occur if other septins provided redundant roles in this process, but in cells transfected with GFP-BD3, all of the septins in the cell are aggregated together and therefore unavailable (unpublished observations and Joberty et al., 2001).
Septins have also been implicated in regulating the stability of the microtubule network by binding to the microtubule-stabilizing protein MAP4 (Kremer et al., 2005), and microtubule dynamics have been implicated in phagocytosis (Araki, 2006; Khandani et al., 2007). However, we were unable to see changes in microtubule clustering, MTOC reorientation toward the phagosome, or changes in the levels of acetylated tubulin following SEPT2 knockdown or BD3 transfection (data not shown), as would be expected if septins regulated microtubule dynamics during this process. Another possible role of septins in phagocytosis could stem from their ability to anchor multiprotein complexes, as they do at the yeast bud neck during cytokinesis (Gladfelter et al., 2001). In mammalian cells the recruitment of filamentous septins may aid in recruiting other associated multiprotein complexes (Joo et al., 2007) to forming phagosomes on the plasma membrane and disruption of filaments would diminish the efficiency of the phagosomal machinery of the cell. Evidence suggests that mammalian septins also participate in vesicle trafficking (Hsu et al., 1998; Beites et al., 2001), a process important for efficient phagosome formation (Bajno et al., 2000; Niedergang et al., 2003). It will therefore be interesting to determine whether depletion of septins in phagocytic cells inhibits membrane/vesicle trafficking during FcR mediated phagocytosis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: William S. Trimble (wtrimble{at}sickkids.ca)
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