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Department of Life Science and Molecular Disease Research Center, the Center for Distributed Sensor Network, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Among the accessory proteins, dynamin is a main regulator of membrane trafficking at the cell surface. It appears to function in part as a mechanochemical enzyme that catalyzes the budding of vesicles from the plasma membrane (Stowell et al. 1999; Danino & Hinshaw 2001; Song & Schmid 2003). It also interacts with several src homology 3 (SH3) domain-containing proteins (Intersectin, endophilin, Abp1 and cortactin) via its proline-rich domain (PRD), thereby integrating them into the endocytic pathway (Ringstad et al. 1997; Roos & Kelly 1998; Simpson et al. 1999; Qualmann & Kelly 2000; Kessels et al. 2001; Cao et al. 2003). Syndapins (PACSIN) have also been shown to bind synaptic dynamin via their C-terminal SH3 domains and to interact with N-WASP, an activator of Arp2/3. N-WASP has been implicated in endocytosis by virtue of its interaction via a PRD with the SH3 domain of syndapin and has been shown to strongly stimulate actin filament nucleation (Qualmann et al. 1999; Kessels & Qualmann 2002). Moreover, several actin-binding or regulatory proteins associate with endocytic accessory proteins, thereby linking the actin cytoskeleton and endocytic processes, which is essential, as actin forms the comet tails that provide the force needed to propel endocytic vesicles (Merrifield et al. 1999). The way in which these molecules are spatially and temporally regulated during these complex interactions remains unclear, however.
We previously showed that SPIN90 interacts with Nck, which is capable of enhancing actin polymerization via the N-WASP pathway (Rivero-Lezcano et al. 1995; Rohatgi et al. 2001). SPIN90 is also known to act on the actin cytoskeleton, playing a key role during myofibril and sarcomere assembly, and to be a component of protein complexes that stabilize cell adhesion at focal contacts (Lim et al. 2001, 2003). In addition, DIP (mDia interacting protein), another name for SPIN90, reportedly regulates Rho GTPase activity (Rho and Rac) and cell adhesion by interacting with Grb2 and Src, and might be involved in regulating the initial steps of cell movement following stimulation of integrin or growth factor receptors (Satoh & Tominaga 2001; Meng et al. 2004). WISH, which is a N-WASP-binding protein, appears to be a mouse ortholog of SPIN90 that enhances N-WASP-dependent and -independent Arp2/3 complex activation, resulting in rapid actin polymerization (Fukuoka et al. 2001). Thus, SPIN90 is able to play a variety of roles during rearrangement of the actin cytoskeleton. Furthermore, we recently characterized that SPIN90 associated with dynamin1 in hippocampal neuron (Kim et al. 2006). Here we show that SPIN90 also participates in receptor-mediated endocytic pathway in fibroblasts.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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SPIN90 contains an SH3 domain and a PRD, which are known to mediate protein–protein interactions. With that in mind, we used a yeast two-hybrid system with full-length SPIN90 as bait to screen a rat brain cDNA library and were able to identify syndapin1 (PACSIN1) as a SPIN90 binding protein (Fig. 1A). That syndapin1 binds directly to SPIN90 was then confirmed in GST pull-down assays using a GST-SPIN90 fusion protein and in vitro-translated syndapin1 or, conversely, GST-syndapin1 and in vitro-translated SPIN90 (Fig. 1B,C). Syndapin1, a synaptic dynamin-binding protein, is specifically expressed in brain, though other isoforms (syndapin2-l and syndapin2-s) are ubiquitously expressed. To identify more precisely the SPIN90 domain that interacts with syndapin1, we carried out in vitro binding assays using the PRD of SPIN90 (SPIN90-PRD) and PRD-deleted SPIN90 (SPIN90-
PRD) with the SH3 domain of syndapin1 (Syn1-SH3) and SH3-deleted syndapin1 (Syn1-SH3). Whereas SPIN90-PRD strongly interacted with Syn1-SH3, as well as with syndapin isoforms (syndapin2-l and 2-s), SPIN90-PRD did not (Fig. 1C), indicating the binding to be dependent on the PRD and SH3 domain. This was noteworthy in part because syndapin1 is known to also interact with dynamin and N-WASP via its SH3 domain, and two other syndapin isoforms (2-l and 2-s) reportedly participate in both endocytosis and actin organization (Qualmann et al. 1999; Modregger et al. 2000; Qualmann & Kelly 2000; Kessels & Qualmann 2004).
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The interaction of SPIN90 with syndapins in vivo was assessed using co-immunoprecipitation assays with affinity-purified anti-SPIN90, anti-syndapin1 or anti-syndapin2-1 antibodies. Co-immunoprecipitation using anti-syndapin antibodies with lysates of rat hippocampal neurons (14 day-old) and COS-7 cells showed that SPIN90 associates with syndapin1 immune complexes from hippocampal neurons and with syndapin2-l in COS-7 cells (Fig. 2A). During clathrin-mediated endocytosis, coated pits on the plasma membrane are formed by the assembly of cytosolic coat proteins, after which these coated pits invaginate and bud to form endocytic vesicles (Slepnev & De Camilli 2000). Numerous endocytic accessory proteins participate in this process. Whether SPIN90 participates in clathrin-mediated endocytosis in vivo by association with other endocytic proteins was determined using co-immunoprecipitation assays carried out. Clathrin, but not amphiphysin, was found within SPIN90-immune complexes from hippocampal neurons (Fig. 2A), and an in vitro binding assay determined that SPIN90 is indirectly associated with clathrin (data not shown). Therefore, its association with endocytic proteins (e.g. syndapins and clathrin) suggests SPIN90 may be present in clathrin-mediated endocytic complexes and participate in such clathrin-mediated endocytic processes such as formation of clathrin coated vesicles (CCVs), invagination and fission.
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We next used immunofluorescence imaging to evaluate the distribution of SPIN90 within cells and its co-localization with other proteins. In primary cultures of hippocampal neurons, immunofluorescence imaging showed that SPIN90 co-localizes with syndapin1 at small punctuate structures within the cell body, neurites and presynaptic terminals (Fig. 2B). In addition, SPIN90 was scattered throughout the cytoplasm in COS-7 cells and co-localized with syndapin 2-l and clathrin as punctuate or small tubular vesicular structures (Fig. 2C,D). It thus appears that SPIN90 is present in complexes with endocytic proteins, including syndapins and clathrin, suggesting it is a component of endocytic pathways.
SPIN90 participates in clathrin-mediated endocytosis
Over-expression of endocytic proteins such as amphiphysin and endophilin inhibits receptor-mediated endocytosis through inappropriate interaction with other endocytic proteins (Wigge et al. 1997; Gad et al. 2000). Bearing that in mind, we assessed the participation of SPIN90 in clathrin-mediated endocytosis by over-expressing it in COS-7 cells. Once we had confirmed that the various HA-tagged SPIN90 deletion constructs were well expressed (Fig. 3A,B), we transiently transfected cells with each of the HA-tagged SPIN90 variants and then assayed uptake of Texas Red-labeled transferrin for 15 min as an index of endocytosis. We found that over-expression of SPIN90-full-length in COS-7 cells significantly inhibited endocytosis, as evidenced by a decrease in transferrin uptake (44.27 ± 9.49% of control at 15 min; Fig. 3C,D). Transferrin uptake was also significantly diminished (38.70 ± 3.73% at 15 min) in cells over-expressing the N-terminus of SPIN90 (SPIN90-N-term: amino acids 1-279, including SH3 and PRD), but was little affected (85.09 ± 5.1% at 15 min) by over-expression of the C-terminus (SPIN90-C-term: amino acids 280-722, SH3- and PRD-deleted) (Fig. 3C,D). Interestingly, over-expression of SPIN90-PRD, which mediates binding to syndapin1, significantly interfered with endocytosis (27.17 ± 4.57% at 15 min). This finding was confirmed by incubating cells transfected with various SPIN90 constructs with biotinylated transferrin and then detecting the internalized transferrin using HRP-conjugated NeutrAvidin. As shown in Fig. 3E, levels of transferrin were substantially diminished in the lysates of cells expressing SPIN90-PRD, which is consistent with the absence of Texas Red-labeled transferrin in the images of cells expressing SPIN90-PRD seen in Fig. 3C. Syndapin also interacts with other proteins containing a PRD, such as dynamin and N-WASP, thereby integrating them into the endocytic process. We next tested whether the interaction of SPIN90-PRD with syndapin interferes with the interaction between syndapin and other PRD-containing proteins (e.g. dynamin). Using competition and co-immunoprecipitation assays, we found that over-expression of SPIN90-PRD dose-dependently interfered with the interaction between syndapin and SPIN90 or dynamin2 in COS-7 cells (Fig. 4A) and that syndapin in SPIN90-PRD transfected cells was, in part, recruited to SPIN90-PRD localized regions (Fig. 4C). However, it had no effect on the cellular distribution of other endocytic components such as clathrin and AP2 (Fig. 4B). These results suggest that SPIN90 participates in the endocytic pathway through the interaction of its PRD and the SH3 domain of syndapin. Thus, SPIN90 and syndapin may act in concert to regulate clathrin-mediated endocytosis.
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To verify whether the observed inhibition of endocytosis in COS-7 cells over-expressing SPIN90-PRD was due to an interruption in syndapin function, we attempted to rescue endocytosis by co-expressing syndapins. Microscopic examination and biochemical assays each revealed that over-expression of SPIN90-PRD or syndapin (syndapin1, syndapin2-l or syndapin2-s) strongly inhibited transferrin uptake (Figs 3 and 5A–C, Modregger et al. 2000), but that if syndapins were co-transfected with SPIN90-PRD, uptake of transferrin was restored to a level comparable to that seen in control cells (Fig. 5A–C).
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Given that over-expression of SPIN90-PRD inhibited endocytosis, we next examined the effect on endocytosis of depleting or knocking down SPIN90. The involvement of endogenous SPIN90 in endocytosis was further confirmed by depleting the protein in COS-7 cells by microinjecting rabbit anti-SPIN90 antibody (Fab fragments), which reduced endocytosis of transferrin to levels similar to those seen in knockdown cells (45.18 ± 3.95% at 15 min). Cells microinjected with normal IgG (Fab fragment) showed transferrin internalization at levels similar to non-injected cells (87.10 ± 2.44% at 15 min) (Fig. 5D,E). For SPIN90 knockdown experiments, five siRNAs targeting different sequences (Fig. 6A) were designed using siRNA Wizard, online accessible software (http://www.sirnawizard.com). Immunoblot analysis showed that three of the siRNAs (si-824, si-1293 and si-1731) effectively reduced endogenous SPIN90 expression, whereas two (si-1666 and si-1735) were ineffective (Fig. 6B) (Kim et al. 2006). In addition, we also found that co-transfecting COS-7 cells with HA-tagged wild-type SPIN90 and si-824, si-1293 or si-1731 efficiently reduced exogenous HA-SPIN90 expression (Fig. 6C). In a parallel experiment, expression of HA-SPIN90 constructs resistant to si-824, si-1293 or si-1731 (see Experimental procedures, Fig. 6D) was unaffected by the three. In cells loaded with Texas Red-labeled transferrin, SPIN90 knockdown using si-824, si-1293 or si-1731 significantly reduced transferrin internalization (si-824, 45.44 ± 2.7%; si-1293, 37.87 ± 0.7%; and si-1731, 40.06 ± 0.6% at 15 min, Fig. 6E) and the effect was reversed by transfection of siRNA-resistant SPIN90 mutants (Fig. 6F,G). By contrast, transferrin uptake was unaffected by Mock (100.00 ± 4.0%), si-1666 (94.34 ± 3.7%) or si-1735 (98.86 ± 1.7%) at 15 min (Fig. 6E,F).
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Internalized components of endocytosis eventually bud from the plasma membrane to form endosomal vesicles, the transport of which is regulated by Rab proteins and their effector proteins. For instance, activated Rab5 is important for the fusion of vesicles with early endosomes (sorting endosomes), while the docking protein EEA1 (early endosome antigen1), a Rab5 effector, is necessary for endosomal membrane fusion activity. To test whether SPIN90 co-localizes with endocytic early endosomes, we fluorescently labeled Rab5 and EEA1 along with SPIN90 and examined their distributions in COS-7 cells. Immunohistochemical analysis revealed that Rab5 and EEA1 partially co-localize or closely associate with SPIN90 (Fig. 7A,B). In fact, SPIN90 was found in association with Rab5 in the same confocal planes through the cells (Fig. 7D). Furthermore, SPIN90 was also found in early endosomes containing internalized FITC-conjugated dextran (Fig. 7C), suggesting that SPIN90 does indeed localize to early endocytic vesicles.
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Vesicle formation and movement is driven by actin dynamics through the activation of various actin regulatory proteins. For instance, dynamin is enriched at the interface between moving endosomes and actin comet tails in cells over-expressing PI(4)P 5-kinases (Lee & De Camilli 2002; Orth et al. 2002; Orth & McNiven 2003). PI(4,5)P2 (Phosphatidylinositol 4, 5-bisphosphate), which is synthesized by PI(4)P 5-kinases, stimulates actin polymerization around membrane vesicles to generate a comet. In an earlier report, DIP (Meng et al. 2004), another name for SPIN90, and WISH (Fukuoka et al. 2001), a mouse ortholog of SPIN90, were shown to play critical roles in actin assembly and reorganization. To determine whether SPIN90 is also present within endosomal vesicles and assists in vesicular trafficking in living cells, COS-7 cells were transfected with GFP-tagged SPIN90 and type I PI(4)P5K, which enhances vesicle formation and actin comet tail formation. After transfection for 18–24 h, we recorded time-lapse sequences of GFP-SPIN90 in living cells. This enabled us to directly observe GFP-SPIN90 as white dots or short tails tagged to the moving endosomal vesicles (Fig. 8A). SPIN90-tagged endosomal vesicles randomly and rapidly moved within the cytoplasm, and similar actin comet tails were also seen in BHK cells (Kim et al. 2006), which suggests SPIN90 is present at moving endosomes. To verify the presence of endocytic vesicles in moving endosomes, COS-7 cells co-transfected with GFP-tagged SPIN90 and type I PI(4)P5K were incubated with Texas Red-EGF (another endocytic marker). Subsequent microscopic analysis clearly demonstrated that SPIN90 localizes in the early endocytic vesicles containing internalized Texas Red-EGF (Fig. 8B), further supporting the idea that SPIN90 plays an important role in the endocytic pathway.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Clathrin-mediated endocytosis is driven by the assembly of clathrin and AP2 complexes and involves a number of accessory proteins that are spatially regulated via their conserved SH3 domains, EH domains (Eps15 homology domain), PRDs and NPF motifs. Several containing SH3 domains (amphiphysin, intersection, endophilin and syndapins) appear to participate via interaction with PRD-containing proteins (dynamin, synaptojanin, and N-WASP) (Simpson et al. 1999; Slepnev & De Camilli 2000). In the present study, we found that syndapin directly interacts with the PRD of SPIN90 via its SH3 domain in vitro and in vivo, though another SH3-containing protein (amphiphysin) did not (Fig. 2A). Moreover, yeast two-hybrid screening did not reveal any other SH3-containing endocytic proteins that interact with SPIN90. In that context, the earlier finding that the syndapin SH3 domain preferentially inhibits a late step leading to CCV budding prompts us to speculate that association between SPIN90 and syndapin is required for CCV fission (Simpson et al. 1999; Kessels & Qualmann 2004). The functionality of endocytic proteins in clathrin-mediated endocytosis requires the appropriate association and dissociation of the proteins involved (Slepnev & De Camilli 2000). Consistent with previous studies, we found that SPIN90 associates with syndapin during endocytosis: over-expression of SPIN90-PRD significantly inhibited transferrin uptake, whereas co-expression of SPIN90-PRD with syndapins restored transferrin uptake to control levels.
It is known that over-expression of endocytic proteins such as amphiphysin and endophilin inhibits clathrin-mediated endocytosis through inappropriate interaction with other endocytic proteins (Wigge et al. 1997; Gad et al. 2000). In that regard, over-expressed SPIN90-PRD probably interferes with syndapin activity similarly, through inappropriate binding. For example, over-expression of SPIN90-PRD interfered, in a dose-dependent manner, with the interaction between syndapin and SPIN90 or dynamin2 in COS-7 cells. Our finding that syndapin was recruited to regions containing SPIN90-PRD within the cells further supports the idea that the interaction of SPIN90 with syndapin is important in endocytic pathway.
Restoration of transferrin uptake by co-expression of SPIN90 and syndapin also is consistent with the idea that the interaction of SPIN90 with syndapin plays a pivotal role in endocytosis, as are our findings that depleting the endogenous protein by microinjecting anti-SPIN90 antibody or knocking down endogenous expression of SPIN90 through RNA interference inhibited endocytic uptake of transferrin. Consistent with our results, the recent report that acute interference with syndapin protein complexes by anti-syndapin antibodies dose-dependently impairs endocytosis (Braun et al. 2005).
Rab proteins and their effectors are critical components participating in endosome formation. They regulate membrane budding to form vesicles and cytoskeletal transport and provide specificity for vesicle docking and fusion (Kurzchalia et al. 1992; Kato et al. 1996; Ohya et al. 1998; Schimmoller et al. 1998; Ungermann et al. 1998; Mohrmann & van der Sluijs 1999). The Rab5 effector EEA1 coordinates the docking of incoming vesicles and SNARE-mediated fusion. Our immunohistochemical analysis shows that EEA1 and Rab5 partially co-localize or associate early (2 min) with SPIN90, as does FITC-dextran, which together suggest SPIN90 may play a role during early endosomal vesicle transport. Additionally, it is interesting to note that GFP-SPIN90 was found in early endocytic vesicles containing endocytic cargo protein (Texas Red-EGF).
The actin cytoskeleton provides the force required to detach newly formed vesicles from the plasma membrane and then propel them through the cytosol (Merrifield et al. 1999), and the movement of the intracellular vesicles (i.e. endosomes) is driven by actin polymerization mediated by Arp2/3 complex (Taunton et al. 2000). Ectopically expressed dynamins act with PI(4)P5K to form actin comet tails, which provide vesicle movement. N-WASP, an activator of Ar2/3 complex, is itself activated by PI(4,5)P2 and then recruited to the vesicle surface associated with the actin comet tail. SPIN90 interacts with various actin regulatory proteins, including WASP and ßPIX. For instance, DIP (another name for SPIN90) is involved in EGF-stimulated cell motility through Src kinase-dependent feedback modulation of Rho GTPase, as well as in the regulation of Rho and Rac activity (Meng et al. 2004). In addition, WISH (a mouse SPIN90 ortholog) induces N-WASP-dependent and -independent Arp2/3 complex activation (Fukuoka et al. 2001) and SPIN90 interacts with Arp2/3 complex to mediate formation of lamellipodia and actin comet tails (Kim et al. 2006). The time-lapse video shown in Fig. 8 reveals that SPIN90 is present moving vesicles in living cells, which suggests SPIN90 is involved not only in endocytosis but also in the trafficking of the endosomal vesicles. It may be that SPIN90 is able to link the endocytic pathway with the actin cytoskeleton, but that awaits further investigation.
Recent studies have also focused on the temporal regulation of endocytic proteins by kinases such as AAK1 and cdk5 (Turner et al. 1999; Conner & Schmid 2002; Sekiya-Kawasaki et al. 2003). In that regard, SPIN90 is phosphorylated by ERK1 upon cell adhesion and by PDGF upon cell activation (Lim et al. 2003). This phosphorylation may in turn facilitate the formation of membrane ruffles in association with actin regulatory proteins (dynamin, WASP, ßPIX and Nck). Ca2+/calmodulin-dependent protein kinase II
(CaMKII) was also identified as potentially able to phosphorylate SPIN90. That CaMKII phosphorylates and thus regulates several synaptic trafficking molecules suggests SPIN90 phosphorylation by CaMKII may be closely related to its regulation of endocytosis (Turner et al. 1999). That said, further study will be required to define precisely the mechanisms that govern the activities of SPIN90 and the relevant kinases during their regulation of endocytosis and/or actin reorganization.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Various SPIN90 constructs were previously described (Lim et al. 2003). Syndapin1 (amino acids 1-441), syndapin2-l (amino acid 1-488), syndapin2-s (amino acid 1-447) were amplified by PCR using the following primers from a rat brain MATCHMAKER cDNA library in pACT2 (BD Clontech, Palo Alto, CA, USA): syndapin1, 5'-CCTACAGGTACCCCATGTCTGGCC-3' (forward) and 5'-GTGGGCGAATTCGCTATATAGCCTC-3' (reverse); syndapin2-l and syndapin2-s, 5'- TTCTCTGGATCCATGTCTGTCACC-3' (forward) and 5'-GCCCATCTCGAGTCACTGGATAGC-3' (reverse). All PCR products were confirmed by full sequencing analysis and then syndapin1, syndapin2-l, syndapin2-s, syndapin1 SH3 (amino acids 1-381), and syndapin1 SH3 domain (amino acids 382-441) were subcloned, in frame, into pGEX4T-1 vector, and His C pcDNA 3.1 vector. Three constructs resistant to si-824, si-1293 and si-1731 were generated using Quick Change Site-Directed mutagenesis (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. Three regions, GTGGCAACTGGT, TGCAAGAGAAAC and AAACACGCCAAT, corresponding to nucleotide positions 826-838, 1297-1309 and 1735-1747, were mutated to GTCGCTACAGGA, TGTAAAAGGAAT and AAGCATGCGAAC, leaving the amino acid sequence unchanged.
Antibodies
Anti-SPIN90 and Anti-Nck was previously described (Lim et al. 2003; Kim et al. 2006) and anti-syndapin1 polyclonal antibodies were generated in both rabbits and mice immunized with purified GST-syn1 SH3 (amino acids 382-441). The antibodies were affinity-purified using the same GST fusion proteins that served as the immunogen. Anti-clathrin (X-22) and anti--adaptin antibodies were obtained from Affinity BioReagents (Golden, CO, USA). Anti-GAL4 DB, anti-GFP, anti-syndapin2-1 (PACSIN2) and anti-dynamin2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Upstate (Charlottesville, VA, USA). Anti-Rab5 and anti-EEA1 were from BD transduction (San Jose, CA, USA). Anti-amphiphysin monoclonal antibody was from Laboratory Frontier (South Korea, Seoul). Anti-HA tag epitope monoclonal antibody (16B12) and anti-HisG were from Covance (Berkeley, CA, USA) and Invitrogen (Carlsbad, CA, USA).
Yeast two-hybrid screening
To construct the bait for yeast two-hybrid screening, a cDNA clone encoding the full SPIN90 gene (1-2169 bp) was inserted into pGBKT7/GAL4 DNA binding vector. Expression of the bait in yeast (AH109) was verified by immunoblotting using monoclonal anti-GAL4 DB or anti-SPIN90 antibodies. A rat brain cDNA library in the pACT2/GAL4 AD vector was purchased from Clontech and amplified according to the manufacturer's instructions, after which two-hybrid screening was carried out according to the Clontech MATCHMAKER Two-Hybrid System manual using the lithium acetate (LiAc) method to prepare competent yeast cells. AH109 cells were co-transformed with pGBKT7/SPIN90 full-length and pACT2/GAL4 AD library fusion constructs. Cells were first plated and selected on double negative synthetic dropout (SD/Trp, Leu minus) media plates. The resultant colonies were then selected secondarily on quadruple negative SD/Trp, Leu, Ade, His minus plates containing 3 mM 3-AT. Colonies were allowed to grow at 30 °C for 3–5 days. The positive interaction between two proteins was revealed by the appearance of large colonies on SD/Trp, Leu, Ade, and His minus plates. To remove false positives, surviving clones were retransformed using bait/syndapin1 or mock vector/syndapin1. The ingredients for yeast media were purchased from DIFCO and Clontech.
GST-pull down assays and immunoprecipitation
For GST pull-down assays, GST-fused recombinant proteins, full-length SPIN90, syndapin1, syndapin2-l, syndapin2-s and the syndapin1 SH3 domain were over-expressed in Escherichia coli BL21 (DE3) and immobilized on glutathione-Sepharose beads according to the manufacturer's procedure. Full-length SPIN90, SPIN90-PRD, SPIN90-PRD, full-length syndapin1, and clathrin heavy chain (CHC), light chain a and b (CLCa and CLCb) were translated in vitro using a TNT T7-coupled reticulocyte lysate system (Promega, Madison, WI, USA). The [35S]-radiolabelled products were incubated overnight at 4 °C with purified GST or GST-fused proteins immobilized on glutathione-Sepharose beads in binding buffer (20 mM Tris-HCl at pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.2% Nonidet P-40, 1 mM PMSF) supplemented with a mixture of protease inhibitors. Bound proteins were subjected to SDS-PAGE and then visualized by autoradiography.
For co-immunoprecipitation assays, cultured cells were extracted for 1 h at 4 °C in modified RIPA buffer (50 mM Tris-HCl at pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF and 1 mM Na3VO4) supplemented with protease inhibitors. The resultant lysates were immunoprecipitated using antibodies against syndapins or SPIN90, after which the immunoprecipitates were incubated for 4 h at 4 °C with protein A-sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and then subjected to SDS-PAGE and immunoblotted with anti-SPIN90, anti-clathrin, or anti-amphiphysin antibodies.
Cell culture, transfection and immunohistochemistry
COS-7 and 293T cells were all grown in DMEM supplemented with 10% FBS and antibiotics at 37 °C in 5% CO2. Primary rat hippocampal neurons were dissected and plated on to poly D-lysine coated coverslips and then maintained for 14 days in neurobasal medium supplemented with 2% B-27. For transfection, cells were plated on glass coverslips (12 mm or 18 mm) or 35 mm tissue culture dishes for 24 h and transfected using Lipofectamine with Plus reagent according to the manufacturer's instructions. For subsequent immunofluorescence imaging, cells were washed 3 times with PBS containing 1 mM CaCl2 and 1 mM MgCl2, fixed for 10 min in 3.5% or 4.0% freshly paraformaldehyde (Sigma Chemical Co.), and then permeabilized for 10 min in 0.2% Triton X-100. Once permeabilized, the cells were incubated at room temperature with primary antibodies for 1 h and then washed 3 times with PBS for 10 min. Fluorophore-conjugated secondary antibodies were applied for 1 h at room temperature.
To label endocytic vesicles, cells were incubated with Texas Red-EGF (1 µg/mL; Molecular Probes) for 10 min, after which they were fixed and processed. The processed cells were washed 3 times and mounted with Vectashield® mount medium (Vector Laboratories, Burlingame, CA, USA). Immunostaining was visualized using a Leica DMRBE microscope (Leica Microsystems AG, Wetzlar, Germany) equipped with a 63x (1.4 NA) oil objective and FITC- or Texas Red-optimized filter sets (Omega® Optical Inc, Brattleboro, VT, USA). Images were acquired using a CoolSNAPTMfx CCD camera driven by MetaMorph imaging software (Universal Imaging Co, Downingtown, PA, USA) and processed by using Adobe Photoshop 7.0.
Transferrin uptake and quantification
Immunohistochemical analysis of transferrin uptake via endocytosis was carried out as previously described (Qualmann & Kelly 2000). Briefly, using Lipofectamine with Plus reagent (Invitrogen), COS-7 cells were transiently transfected with the appropriate SPIN90 cDNA constructs or with syndapin1, -2-l or -2-s. Following transfection, the cells were incubated for 36–48 h and then serum starved for 2 h before incubation for 15 min at 37 °C with 20 µg/mL Texas Red-labeled transferrin. Surface-bound ligand was removed by a brief acid wash with deferoxamine mesylate (pH 5.3) in 150 mM NaCl and 2 mM CaCl2, after which the cells were fixed and prepared for immunofluorescence microscopy. For rescue assays, cells were co-transfected with HA-tagged SPIN90-PRD and His-tagged syndapin constructs. Transferrin internalization was quantified by image analysis in which the intensity of transferrin signals in cell images were measured after background subtraction using MetaMorph imaging software. Thereafter, the transferrin signals were normalized to those in control images and were expressed as percentages of control after processing using Origin 6.1 (OriginLab, Northampton, MA, USA).
Biochemical assays of transferrin uptake were carried out as previously described (Engqvist-Goldstein et al. 2004) with slight modification. 293T cells were plated in 6-well plates and transfected with the appropriate constructs. After incubation, the cells were washed with serum-free DMEM and serum starved for 2 h in the same medium, after which they were incubated for 15 min at 37 °C in medium containing biotin-transferrin (2.5 µg/mL; Molecular Probes) and unlabeled transferrin (500 µg/mL; Sigma). To detect internalized transferrin, the cells were placed on ice and washed twice with ice-cold PBS, after which surface-bound transferrin was stripped away by incubating the cells for 10 min in ice-cold deferoxamine mesylate (pH 5.3) in 150 mM NaCl and 2 mM CaCl2. The cells were then washed twice with ice-cold PBS and lyzed with lysis buffer [1% SDS, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM Tris-HCl (pH 7.4), 1 mM PMSF, 10 mM leupeptin, 1.5 mM pepstatin, and 1 mM aprotinin]. Protein concentrations were measured using a bicinchoninic acid (BCA) Protein Assay Reagent kit (Pierce, Rockford, IL, USA). Constant amounts of lysate were subjected by SDS-PAGE. Internalized transferrin was detected using HRP-conjugated NeutrAvidin‘ (Molecular Probes) with Enhanced Chemiluminescence (ECL) reagents (Amersham Biosciences). In some cases, blots were stripped by heating to 60 °C for 30 min in stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and reprobed.
RNA interference
SPIN90 siRNAs were previously described (Kim et al. 2006). SPIN90 siRNAs were cloned into the Acc65I-HindIII sites of the psiRNA-hH1GFPzeo G2 vector (Invivogen, San Diego, CA, USA), a siRNA expression vector containing a GFP gene driven by a separate promoter for tracking transfection efficiency. COS-7 cells were transfected for 72 h with SPIN90-specific siRNAs alone or together with HA-SPIN90 or siRNA resistant mutants using lipofectamine with Plus reagent (Invitrogen). Protein expression was confirmed by immunoblotting using anti-SPIN90 or anti-HA antibodies.
Confocal microscopy
Confocal images were acquired using a Leica TCS SP2 confocal laser scanning unit (Wetzler, Germany) equipped with Ar/ArKr and He-Ne lasers and attached to a Leica DMIRE2 inverted microscope. The cells were observed using a 63x objective lens (1.4 NA). Images were acquired and were processed using Leica confocal LCS software.
Microinjection
To inject antibody, Fab fragments of normal rabbit IgG (sigma) and rabbit anti-SPIN90 antibodies were prepared using an ImmunoPure, Fab Preparation Kit according to the manufacture's instructions (Pierce). Then using a Transjector 5246 and Micromanipulator 5171 (Eppendorf, Hamburg, Germany) attached to a Leica DMIRB inverted microscope, cells grown on glass coverslips (18 mm) for 24 h were microinjected with 10 µL of normal rabbit IgG (3.4 mg/mL) or anti-SPIN90 antibodies (3.0 mg/mL) in microinjection buffer (10 mM KH2PO4, pH 7.2, 75 mM KCl). After injection, the cells were allowed to recover in 5% CO2 at 37 °C for 4 h and then used for endocytosis assays. To observe the distribution of antibodies, the injected cells were incubated with FITC-conjugated secondary antibody.
Live cell imaging
Cells were initially grown on 18 mm glass coverslips and transfected for 24 h with GFP-SPIN90-full-length with PI(4)P5 kinase. Thereafter, they were placed in a parallel plate chamber or in a sealed chamber containing oxygen-depleted (Oxyrase, Mansfield, OH, USA) tyrode solution (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.4, 30 mM glucose). Time-lapse images were acquired using a Leica DMIRB inverted microscope equipped with a 63x (1.4 NA) oil objective (Leica Microsystems AG, Wetzlar, Germany) and a CoolSNAP-HQ CCD camera driven by MetaMorph imaging software. Time-lapse sequences were constructed by acquiring 500 ms exposures with 3- to 5-s intervals using a VMM3 Uniblitz shutter (Vincent Associates, Rochester, NY, USA).
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* Correspondence: E-mail: wksong{at}gist.ac.kr
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Received: 18 April 2006
Accepted: 20 June 2006
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