Correspondence to Alice Dautry-Varsat: adautry{at}pasteur.fr
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Introduction |
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Glycosylphosphatidylinositol (GPI)-anchored proteins, the autocrine motility factor, the TGF-ß receptor, the ß chain of interleukin (IL)-2 receptor, toxins, but also viruses, are examples of the growing number of markers taken up by clathrin-independent endocytosis (for reviews see Gesbert et al., 2003; Nabi and Le, 2003; Nichols, 2003; Pelkmans and Helenius, 2003). These markers are not found in clathrin-coated pits; instead they are associated with membrane lipid microdomains enriched in cholesterol and glycosphingolipids, also called "rafts," caveolae being a subset of these domains that contain caveolin (Nabi and Le, 2003). The GTPase dynamin seems to play an essential role in most clathrin-independent internalization examples reported, but its function in these processes is unclear. However, dynamin is not required for all endocytic pathways, in particular pinocytosis of GPI-anchored proteins can be dynamin independent (Mayor and Riezman, 2004). Very little is known about the role of actin dynamics in clathrin-independent endocytosis. It was shown, in the case of Simian Virus 40 uptake, that dynamin and actin were recruited to the virus-loaded caveolae (Pelkmans et al., 2002). In addition, the requirements for activated GTPases of the Rho family in the uptake of GPI-anchored protein and in the IL-2 receptor ß chain entry are in favor of a role of the actin cytoskeleton in clathrin-independent mechanisms (Lamaze et al., 2001; Sabharanjak et al., 2002). More generally, due to the lack of molecular characterization of clathrin-independent endocytosis, these pathways are far from being understood.
The common cytokine receptor c belongs to the type I cytokine receptor family that lack intrinsic kinase activity but recruit molecules that transduce the signal to the cell. The
c chain is shared by IL-2, -4, -7, -9, -15, and -21 receptors (Schluns and Lefrancois, 2003). Thus, the
c receptor plays a major role in lymphocyte proliferation and differentiation, leading when mutated to X-linked severe combined immunodeficiency (Schluns and Lefrancois, 2003). This receptor is rapidly internalized and reaches early endosomes (Hémar et al., 1995). Then,
c is targeted to late endosomes and lysosomes where it is degraded. Therefore,
c endocytosis leads to its down-modulation and thus is involved in the control of signaling pathways induced by the ILs that use it as a receptor component (Morelon et al., 1996). The
c receptor contains, in its cytosolic tail, sequences that enable its efficient and rapid endocytosis independently of the ligand or of other receptor chains (Morelon and Dautry-Varsat, 1998; Yu et al., 2000). However, the mechanism by which
c receptor is internalized is not known. The lack of a classic clathrin-coated pit localization signal in
c receptor and the clathrin-independent uptake of another cytokine receptor member, the ß chain of IL-2 receptor, suggested that
c endocytosis might be clathrin independent. In this work, we show that indeed the
c receptor endocytosis is independent of AP-2 and clathrin and we analyzed the role of dynamin partners that also interact with F-actin.
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Results |
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Intersectin is a scaffolding protein belonging to this class of dynamin-binding proteins involved in clathrin-dependent endocytosis (Simpson et al., 1999). Intersectin has an Eps15 homology domain that recruits epsin. Epsin binds AP-2, thus linking intersectin to the clathrin-coated pathway (Yamabhai et al., 1998; Sengar et al., 1999). It also contains five SH3 domains binding to dynamin, synaptojanin, and N-WASP, the latter making a link with the actin cytoskeleton as it is a potent activator of the Arp2/3 complex (Hussain et al., 2001). To test the involvement of intersectin in c receptor internalization, cells were cotransfected with
c gene and with a dominant-negative mutant of intersectin composed of its five SH3 domains (IntersecDN; Simpson et al., 1999). Tf and
c receptor uptake were monitored in doubly transfected cells as described in the previous paragraph and their internalization was analyzed by confocal microscopy (Fig. 6 B). As expected, 85% of the transfected cells were deeply affected for Tf uptake. In contrast, no effect was observed for
c receptor endocytosis (Fig. 6 B). Thus, intersectin is a dynamin-binding protein that does not appear to be necessary for the clathrin-independent endocytosis of
c receptor.
The protein syndapin, which binds to the PRD domain of dynamin, has also been shown to regulate clathrin-dependent endocytosis (Qualmann and Kelly, 2000). In addition to dynamin, syndapin also interacts with N-WASP, providing a link with the actin cytoskeleton (Kessels and Qualmann, 2002). To assay for a putative function of syndapin in c receptor uptake, cells were transfected with the dominant-negative mutant of syndapin 2 (Sdp2DN), composed of the SH3 domain of Sdp2 (Qualmann and Kelly, 2000), and endocytosis of the
c receptor was compared with that of Tf. In more than 80% of the cells overexpressing this dominant-negative mutant, Tf internalization was inhibited whereas endocytosis of the
c receptor was not affected (Fig. 6 B). Thus, syndapin is another example of a dynamin partner specifically involved in clathrin-mediated endocytosis.
Another protein, which links the actin cytoskeleton to endocytosis via dynamin, is mAbp1 (Schafer, 2002). This protein binds directly to the PRD domain of dynamin on one hand, and to F-actin on the other hand. mAbp1 is colocalized with dynamin in clathrin-coated pits and is involved in clathrin-mediated endocytosis (Kessels et al., 2001; Mise-Omata et al., 2003). Overexpression of the dominant-negative mutant of mAbp1 (mAbp1DN), composed of the SH3 domain of mAbp1, inhibits clathrin-mediated uptake (Kessels et al., 2001). Therefore, we transfected cells with this dominant-negative mutant and the c gene, and assayed for
c receptor and Tf endocytosis. As previously reported, Tf internalization was inhibited in more than 80% of the cells expressing the mAbp1 mutant. However, endocytosis of
c receptor was not inhibited (Fig. 6 B). Once again,
c receptor endocytosis can be distinguished from clathrin-dependent internalization by the requirement for mAbp1.
Finally, the only other known F-actin binding protein that directly binds to the PRD domain of dynamin is cortactin (Orth and McNiven, 2003). As mAbp1, cortactin contains F-actin binding repeats and a SH3 domain at the COOH terminus that binds dynamin. In addition, it contains an NH2-terminal motif (DDW) that binds directly to Arp3 to stimulate Arp2/3-dependent nucleation (Uruno et al., 2001; Weaver et al., 2002). Microinjection of anti-cortactin antibodies or transfection of the cortactin SH3 domain allowed Cao et al. (2003) to demonstrate that cortactin is necessary for clathrin-mediated endocytosis. Therefore, we investigated its putative role in clathrin-independent uptake. Cells were cotransfected with c gene and with the dominant-negative mutant of cortactin (CortDN), constituted by the SH3 domain of cortactin (Du et al., 1998). The distribution of Tf and of
c receptor were compared by confocal microscopy after allowing internalization of both markers simultaneously for 15 min at 37°C (Fig. 7 A). Tf internalization was inhibited in more than 80% of the cells expressing the cortactin dominant-negative mutant, in agreement with published data (Fig. 7 A). Strikingly, endocytosis of
c receptor was also inhibited in 85% of the cells expressing this mutant (Fig. 7 A). In addition, we used siRNA to decrease cortactin expression within the cells. In cells treated with cortactin siRNA (CortsiRNA), the amount of cortactin was clearly reduced, as shown by Western blot, whereas a control protein, flotillin 2, was not affected (Fig. 8 A). We analyzed the internalization of
c receptor and Tf in siRNA-treated cells by confocal microscopy (Fig. 8 B). As shown in Fig. 8 C, both Tf and
c receptor endocytosis were inhibited to the same extent (in
80% of treated cells). Together, these experiments show that cortactin is essential for clathrin-dependent and -independent endocytosis. Cortactin and mAbp1, which have several common features, differ in their ability to bind directly to Arp3. We asked if the direct interaction of cortactin with Arp3 was important for endocytosis. Cells were cotransfected with
c gene and with the gene encoding cortactin mutated in the Arp3 binding site (CortW22A; Schafer et al., 2002). More than 80% of transfected cells were deficient in
c and in Tf internalization, as measured by confocal microscopy analysis and quantification (Fig. 7 B). Thus, cortactin needs to have its Arp3 binding site to participate correctly in these two endocytic pathways. In conclusion, cortactin is the only protein linking dynamin to the cytoskeleton known so far that is involved in both clathrin-dependent and -independent endocytosis.
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Discussion |
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We would like to discuss the specificity of action in internalization processes of each dynamin partner tested in this paper in light of what is currently known in the literature. The lipid PI(4,5)P2 binds to several proteins such as epsin, AP-2, or dynamin and plays a central role in clathrin-mediated endocytosis (Jost et al., 1998; Ford et al., 2002; Rohde et al., 2002). Synaptojanin 2 is a phosphoinositide phosphatase that controls the amount of this lipid in the cell. In addition, synaptojanin 2, via its PRD domain, binds to clathrin-associated proteins endophilin and amphiphysin (Hill et al., 2001; Nemoto et al., 2001). Finally, synaptojanin 2 was shown to play an essential role in the early steps of clathrin-coated pits and vesicles formation (Rusk et al., 2003). Altogether, these findings suggest that synaptojanin is a component of the clathrin machinery. Therefore, our result showing that this phosphatase is not essential for c receptor uptake is not surprising. Similar arguments can explain why intersectin is not necessary for the clathrin-independent endocytosis of
c receptor. Indeed, this protein contains an Eps15 homology domain that interacts with epsin, a coiled-coil region binding to Eps15, and finally five SH3 domains interacting with several proteins including synaptojanin (Engqvist-Goldstein and Drubin, 2003). Thus, intersectin seems to be a specific player of clathrin-dependent endocytosis. However, a recent paper has shown that this protein was involved in caveolae fission in endothelial cells, suggesting that this protein may also be involved in caveolae-dependent internalization (Predescu et al., 2003). The abundance of caveolae in endothelial cells, and their crucial role in transcytosis, coupling endocytosis and exocytosis by rapid and efficient fusion-fission processes, suggested a particular function of caveolae in these cells. This finding might explain the special feature of intersectin in this case.
Syndapin has been involved in clathrin-dependent uptake (Qualmann and Kelly, 2000). In addition to dynamin, syndapin binds also to synaptojanin. Our results show that syndapin 2 is not required for the clathrin-independent endocytic pathway taken by c receptor. Altogether, these findings suggest that syndapin may be a specific component of the clathrin-dependent endocytosis.
Cortactin and mAbp1 have been recently implicated in clathrin-mediated endocytosis (Kessels et al., 2001; Cao et al., 2003). Our study shows that whereas mAbp1 is not required for c receptor uptake, cortactin is involved in this clathrin-independent endocytosis, as shown by using siRNA and cortactin mutants. These two proteins share a lot of common features. Indeed, both proteins bind directly to F-actin and dynamin (Orth and McNiven, 2003). In addition, cortactin and mAbp1 are Src kinase targets and are translocated to the cell periphery by the activation of Rac1 (Weed et al., 1998; Kessels et al., 2000). However, it is noteworthy that the two proteins have several specific partners. In particular, they differ by the presence of an acidic motif binding and activating Arp2/3 complex at the NH2 terminus of cortactin (Weed et al., 2000). Interestingly, we found that a cortactin mutant (CortW22A), which no longer binds Arp3, inhibits clathrin-dependent and -independent endocytosis (Fig. 7 B). Thus, the differential effect of cortactin and mAbp1 on
c receptor endocytosis can be accounted for by their different properties in Arp3 binding. The results obtained with CortDN and CortW22A mutants suggest that both the SH3 domain and the Arp3 binding domain are important for cortactin function, as suggested by in vitro studies (Schafer et al., 2002). Cortactin is the only dynamin partner that has been shown to modulate actin dynamics. Indeed, mutations in cortactin avoiding its binding to Arp2/3 or dynamin decrease the actin dynamics of the cell (Schafer et al., 2002). Also, in vitro studies have shown that the complex formed by cortactin, Arp2/3, and dynamin promotes actin assembly (Schafer et al., 2002). Thus, cortactin constitutes the strongest link between endocytosis and the cytoskeleton. The fact that a cortactin mutant incapable of binding Arp3 affects both clathrin-dependent and -independent endocytosis suggests that the action of cortactin is linked to Arp2/3 complex and to actin polymerization.
Recently, clathrin-dependent endocytosis and caveolae-mediated uptake of Simian Virus 40 have been shown to involve actin polymerization at the site of entry (Merrifield et al., 2002; Pelkmans et al., 2002). We have shown here that actin polymerization is also necessary for clathrin-independent receptor-mediated endocytosis of c receptor. Dynamin seems to play a crucial role in the actin cytoskeleton recruitment during endocytosis. The recent results concerning the clathrin-dependent uptake have allowed the identification of several dynamin-binding proteins that could act at the interface between endocytosis and actin dynamics (Orth and McNiven, 2003). In contrast, nothing was known for clathrin-independent endocytosis. However, the central role of dynamin in clathrin-dependent endocytic processes prompted us to test the role of actin-dynamin interacting partners in the clathrin-independent pathway used by the
c receptor. The majority of the known putative linker proteins seem to be solely involved in clathrin-mediated internalization. Cortactin is the only dynamin partner identified so far that is necessary for
c receptor internalization. Interestingly, the ß chain of IL-2 receptor, another marker of the clathrin-independent pathway (Lamaze et al., 2001), also requires cortactin to be internalized (unpublished data), suggesting a general role of cortactin in clathrin-independent endocytosis. Thus, cortactin is involved in both clathrin-dependent and -independent uptake. Our work points to an ubiquitous and pivotal role of cortactin in endocytosis. We propose a working hypothesis whereby dynamin, cortactin, Arp2/3, and F-actin constitute a core complex that would link endocytosis to actin dynamics (Fig. 9). The other dynamin partners, such as syndapin, intersectin, or mAbp1, specific to one endocytic pathway, could play a role in this dynamic process, in association to the core complex or not (Fig. 9). It is noteworthy that mAbp1, syndapin, and intersectin might be involved in some way in clathrin-independent uptake; if so, their role must be less critical than it is for the clathrin pathway. Future work will focus on the precise action of this complex during the different steps of internalization to characterize further the interface between endocytosis and the cytoskeleton.
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Materials and methods |
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Cells lysis and fractionation
Kit225 cells (5 x 107) were resuspended in 0.5 ml of ice-cold TBS (140 mM NaCl, 20 mM Tris, and 1 mM EDTA, pH 7.4), 0.5% Triton X-100, 1 mM phenylmethylsulfoxide, and 1% of inhibitor cocktail (Sigma-Aldrich). After 20-min incubation on ice, the lysate was passed 10 times through a 26G3/8 needle and centrifuged at 800 g for 10 min at 4°C. The supernatant mixed with an equal volume of 80% sucrose was fractionated on a 365% sucrose density gradient (Lamaze et al., 2001). Finally, proteins from the 10 fractions were precipitated by TCA and one third was loaded on SDS-PAGE and analyzed by Western blot with either goat anti-c receptor (1:1,000, AF284; R&D Systems), mouse anti-rab5 (1:1,000; BD Biosciences), or mouse anti-flotillin 2 (1:2,000; BD Biosciences).
Transfection and siRNA experiments
HeLa cells (4 x 106 cells/point) were cotransfected by electroporation with 10 µg of pRCH and 20 µg of plasmid encoding the various mutants at 900 µF and 200 V (Easyject; Eurogentec). 24 h later, the cells were resuspended in DME containing 6 mM Na butyrate to enhance the expression of the mutants, and 24 h thereafter the cells were analyzed by immunofluorescence. siRNAs were synthesized by Dharmacon, and the siRNA duplexes were prepared according to the manufacturer's instructions to yield a 20-µM final concentration. The siRNA sequence targeting synaptojanin 2 was aacgugaacggaggaaagcagtt (Rusk et al., 2003), the one targeting clathrin heavy chain was taatccaattcgaagaccaat (Motley et al., 2003). Two siRNAs targeting cortactin were cotransfected in Hep2 cells, gacugagaagcaugccucctt (Bougneres et al., 2004) and ggagcauaucaacauacactt. We checked that an irrelevant siRNA had no effect on endocytosis. The cells were plated to 80% confluency in 6-well plates. 10 µl of duplexes siRNA or control (10 µl of TE 1/10: 1 mM Tris, pH 7.4, and 0.1 mM EDTA) were mixed in 42 µl of 250 mM CaCl2 solution and added to the cells. Finally, 42 µl of HBS (50 mM Hepes, 280 mM NaCl, 10 mM KCl, and 1.5 mM Na2HPO4, pH 7.05) were added to the cells. 48 h later, 20 µg of pRCH
DNA were introduced by electroporation in siRNA-transfected cells, together with 10 µl of CHCsiRNA or 10 µl of CortsiRNA duplexes, in experiments using these siRNAs. 24 h later, total protein extracts from 105 cells were loaded on SDS-PAGE and analyzed by Western blot with an anti-clathrin heavy chain mouse antibody (BD Biosciences; 1:1,000) or an anti-synaptojanin 2 rabbit antibody (1:2,000; a gift from M. Symons, Center for Oncology and Cell Biology, Institute for Medical Research at North Shore-LIJ, Manhasset, NY; Malecz et al., 2000) or an anti-cortactin mouse antibody (Upstate Biotechnology; 1:2,000) or an anti-flotillin 2 mouse antibody (BD Biosciences; 1:2,000). The secondary antibodies used were alkaline phosphatase-coupled antirabbit or antimouse antibodies (Pierce Chemical Co.). The Western blots were revealed by ECF (Amersham Biosciences) and quantified using a Storm FluoroImager (Molecular Dynamics).
Endocytosis, immunofluorescence, and confocal microscopy
Endocytosis of c receptor and Tf were performed for 15 min at 37°C as described previously (Morelon and Dautry-Varsat, 1998) using anti-
c receptor Tugh4 (rat antibody; BD Biosciences; 1:50) or MAB284 (mouse antibody; R&D Systems; 1:50) and 50 nM of human iron-loaded Tf conjugated to Cy5 or Cy3 (Lamaze et al., 2001). In Fig. 6 A, HeLa cells were pretreated for 45 min at 37°C with either DMSO (1:1,000) or Jasplakinolide (1µM; Molecular Probes). Immunofluorescence experiments were performed as described previously (Morelon and Dautry-Varsat, 1998) with anti-Xpress (mouse antibody; Invitrogen; 1:500), anti-myc (mouse antibody 9E10, ascites 1:500), anti-flagM5 (mouse antibody; Sigma-Aldrich; 1:100), or anti-cortactin mouse antibody (Upstate Biotechnology; 1:200). Secondary antibodies were Alexa Fluor 488coupled antirat (Molecular Probes; 1:100), fluorescein (FITC)-coupled antimouse IgG (Southern Biotechnology Associates, Inc.; 1:100), or Texas redcoupled antimouse IgG1 (Southern Biotechnology Associates, Inc.; 1:100). Confocal microscopy was performed on a microscope (model TCS4D; Leica) using a 63x objective. Z series of optical sections were acquired at 0.7 µm. Cy5, FITC, and Texas red emissions were collected separately to avoid fluorescence passage from one channel to another. Quantification of Tf and
c receptor uptakes were performed within the same cell and for at least 60 doubly transfected cells in at least three experiments.
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Acknowledgments |
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This work was supported by the Association pour la Recherche sur le Cancer, La Ligue Contre le Cancer, Programme dynamique et réactivité des assemblages biologiques, and Action Concertée Incitative Biologie Cellulaire, Moléculaire et Structurale.
Submitted: 29 June 2004
Accepted: 22 November 2004
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