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Article |
Correspondence to Tomoya Katakai: tkatakai{at}virus.kyoto-u.ac.jp
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Abstract |
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Abbreviations used in this paper: AID, autoinhibitory domain; BIM, bisindolylmaleimide I; CT, COOH-terminal; ERM, ezrin/radixin/moesin; GEF, guanine nucleotide exchange factor; MTOC, microtubule organizing center; NT, NH2-terminal; p-ERM, phosphorylated ERM; ROCK, Rho-associated coiled coilcontaining protein kinase; SDF-1, stromal cellderived factor-1; WT, wild-type.
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Introduction |
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Migrating lymphocytes possess a unique cellular structure called the uropod, a spherical membrane protrusion budding from the rear part of the cell body (Sánchez-Madrid and del Pozo, 1999). The roles of the uropod in lymphocyte migration, as well as the molecular mechanisms organizing this structure, are largely unknown. The finding that several molecules are selectively compartmentalized at the uropod provides some clues to clarify these subjects. For instance, some transmembrane adhesion molecules, including CD43, CD44, intercellular adhesion molecules, and PSGL-1, are concentrated at the uropod (Sánchez-Madrid and del Pozo, 1999). These molecules have a motif that can bind ezrin/radixin/moesin (ERM) proteins at the intracellular part; therefore, it is reasonable to suggest that the ERM proteins are also accumulated in the uropod (Mangeat et al., 1999; Sánchez-Madrid and del Pozo, 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002). ERM proteins act as membranecytoskeleton linkers by binding to the membrane proteins at their NH2-terminal (NT) domains and to F-actin at its COOH-terminal (CT) domain (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Gautreau et al., 2000; Bretscher et al., 2002). The linker function is regulated by the phosphorylation of a conserved threonine residue in the CT domain of each ERM protein. This Thr phosphorylation disrupts the intramolecular interaction between the NT and CT domains, allowing them to bind to membrane proteins and F-actin. However, the roles of ERM proteins and Thr phosphorylation in uropod formation or lymphocyte polarity remain to be elucidated.
In this study, by using a T lymphoma cell line, EL4.G8, that constitutively possesses a clear uropod, we show potential roles of the phosphorylated ERM (p-ERM) proteins not only in uropod formation but also in cell polarization in cooperation with RhoROCK signaling.
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Results |
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The role of Thr567 phosphorylation in the uropod localization of ezrin
To address the details of ezrin localization in the uropod, we transfected plasmids expressing various GFP-tagged mutant forms of ezrin into EL4.G8 cells and obtained multiple stable lines (Fig. 2, A and D). Transfection with the control vector expressing GFP resulted in cytoplasmic and nuclear distribution of this reporter protein (Fig. 2, B and C). In contrast, wild-type (WT) ezrin-GFP was preferentially localized at the peripheral membrane, especially in the uropod rather than the cell body, although a weak signal was also detected in the cytoplasm. A mutant ezrin in which the CT Thr was replaced by Ala (T567A) was distributed diffusely in the cytoplasm. This mutant cannot be phosphorylated at this residue and, therefore, does not undergo the intramolecular NT and CT domain dissociation and mimics the dormant form of ezrin (Gautreau et al., 2000; Coscoy et al., 2002). In marked contrast, another mutant with replacement of this Thr by Asp, T567D, which does not undergo NT and CT domain association and thus mimics the phosphorylated active form with membranecytoskeleton linker activity (Gautreau et al., 2000; Coscoy et al., 2002), exclusively accumulated in the uropod colocalized with CD44 (Fig. 2 B, asterisks). The fluorescent signal of the NT domain fragment tagged with GFP was also detected at the uropod, as well as at the peripheral membrane of the cell body, confirming the requirement of the NT domain for ezrin to localize in the uropod. In contrast, the CT domain fragment of ezrin was accumulated at the leading edge and was well-colocalized with F-actin, but was not abundant in the uropod, indicating that this fragment freely associates with the actin cytoskeleton. Such intracellular localization patterns of ezrin mutants were also observed in another T cell lymphoma line, BW5147, although the cells bearing a uropod in this line are a minor population and the sizes of the uropods are much smaller than in EL4.G8 cells (unpublished data).
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T567D ezrin enhances uropod size
The size of the uropod was markedly enhanced by T567D ezrin, whereas T567A ezrin had little effect on it (Fig. 2 F). Forced expression of WT ezrin only slightly augmented the uropod's size, suggesting that the total capacity for ERM Thr phosphorylation is limited in each cell and that a fraction of ezrin-GFP proteins is incorporated into the uropod membrane. Because NT ezrin transfectants showed a slight diminishment of the incidence of cells carrying the uropod (64% for NT, compared with 82% for the vector or WT) and of the uropod's size (Fig. 2 F), this type of deletion mutant might function in a weakly dominant-negative fashion. Interestingly, F-actin was apparently accumulated at the enlarged uropod in T567D ezrin transfectants, demonstrating that the activated ezrin facilitates the organization of the cortical actin cytoskeleton underneath the uropod membrane (Fig. 2 C, arrows). Thus, Thr567 phosphorylation of ezrin can influence the integrity of the uropod in T lymphocytes.
T567D ezrin induces construction of a plasma membrane polar cap in uropod-less situations
As expected, staurosporine treatment disrupted the uropod as well as the polarized distributions of GFP signal and CD44 in WT ezrin transfectants (Fig. 3 A). Surprisingly, in T567D ezrin transfectants, despite the fact that the typical uropod also disappeared upon treatment with staurosporine, the GFP signal and CD44 clearly accumulated at one pole of the cells to form a capped structure (Fig. 3, A [arrows] and B). A similar cap structure was constructed in BW5147 cells transfected with T567D ezrin and treated with staurosporine (unpublished data). These findings strongly suggest that the T567D ezrin can preserve the plasma membrane polarity even in the uropod-abolished cells. Because polar cap formation was not observed in NT ezrin transfectants, this phenomenon requires the phosphorylated CT domain. In addition, cytochalasin D canceled the induction of polar cap formation by T567D ezrin (Fig. 3, A and B), suggesting that F-actin is also required for this event.
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To confirm the ability of T567D ezrin to induce the polar cap in naturally unpolarized cells, we transfected the construct into the myeloma cell line X63.653, which does not carry a uropod or show specific CD44 (Fig. 3 E) and F-actin localization (not depicted). Strikingly, T567D ezrin induced the construction of the polar cap accompanied by CD44 accumulation in these cells, although the uropod was not induced, whereas WT, NT, or T567D-AB ezrin had no influence on CD44 localization (Fig. 3, E [arrows] and F). These results indicate that X63.653 cells seem to be inactive in the machinery not only for ezrin phosphorylation but also for uropod protrusion, and, hence, T567D ezrin induces only plasma membrane polarization in these cells. Together, these findings suggest that Thr567-phosphorylated ezrin has the ability to organize the plasma membrane polarity in several uropod-less situations, whereas uropod protrusion requires an additional mechanism.
RhoROCK signaling is required for uropod protrusion, but not for polar cap formation
Because several Ser/Thr kinases (including the Rho effector ROCK and PKCs) have been shown to phosphorylate ERM proteins (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002), we next treated EL4.G8 cells with an inhibitor for ROCK (Y-27632) or for PKCs (bisindolylmaleimide I [BIM]). BIM had no remarkable effect on the morphology of the cells (not depicted). In contrast, Y-27632 completely eliminated the uropod, although the CD44/p-ERM polar cap was maintained (Fig. 4, A and B). The preservation of p-ERM was confirmed by Western blotting (Fig. 4 C). The phosphorylation status was also unchanged in Y-27632treated BW5147 cells, despite the fact that the uropod was completely disrupted (unpublished data). These experiments demonstrate that endogenous p-ERM proteins are also capable of constructing the polar cap in uropod-less cells. Likewise, transfectants of WT as well as T567D ezrin exhibited a polar cap but not a uropod under the ROCK-inhibited condition (Fig. 4, A and B). Thus, ERM phosphorylation is independent of ROCK as well as of PKCs in EL4.G8, but ROCK activity is indispensable for the formation of the mature uropod.
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Phosphorylated ezrin interacts with Dbl and induces Rho activation
To address the possibility that p-ERM is involved in RhoROCK signaling, we checked the level of Rho-GDP/GTP exchange activity associated with p-ERM in EL4.G8 cells. Using immunoprecipitated p-ERM protein complex from the cell lysate, the exchange activity was assessed by an in vitro nucleotide exchange reaction for recombinant Rho-GDP, followed by RBD (Rho-binding domain)-GSTmediated pull-down detection of Rho-GTP. Strikingly, we detected a substantial nucleotide exchange activity for Rho in p-ERMcontaining precipitate, whereas no activity was detected using control antibody (Fig. 5 A). When p-ERM was depleted previously from the cell lysate, the exchange activity associated with ezrin was markedly reduced (Fig. 5 D).
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To determine whether Dbl is involved in uropod formation in vivo, we transfected Dbl-AID, a construct for the GFP-tagged autoinhibitory domain (AID) of Dbl that potentially interferes with the GEF activity mediated by Dbl (Bi et al., 2001), into EL4.G8 cells. Compared with the results of control experiments, the percentage of cells exhibiting the uropod in the Dbl-AIDexpressing cells was significantly decreased (Fig. 5 G), suggesting that Dbl plays a substantial role in uropod formation. However, because the inhibitory effect of Dbl-AID is insufficient, it is suggested that the dominant-negative action of this artificial protein fragment might be limited in vivo, or that the other GEFs might associate with p-ERM (Fig. 5, compare the residual GEF activity in E with that in D). Together, these results suggest that phosphorylated open-form ezrin, but not the latent form, potentially functions as an upstream regulator for Rho activation and uropod formation, through its binding to Dbl in EL4.G8 cells.
A polar cap is constructed over the posterior cytoplasm
It is well-established that migrating lymphocytes exhibit unique intracellular polarity, i.e., that the microtubule organizing center (MTOC) and the Golgi apparatus are arranged in the posterior part behind the nucleus, thus revealing a cytoplasmic frontrear axis (Sánchez-Madrid and del Pozo, 1999). MTOC positioning in EL4.G8 cells was rather flexible: 70.5% of cells showed the rear MTOC, whereas in some cells, the MTOC was positioned clearly in the front cytoplasm, near the leading edge (Fig. 6 A). The Golgi apparatus was located in the middle or front part of the cell (in 72.6% of cells; Fig. 6 A), as was revealed by staining with anti-GM130 antibody. These unstable locations of the MTOC or the Golgi apparatus might be caused by the rapid cell cycle in this cell line. Ceramide analogues have been reported to label the Golgi apparatus (Pagano et al., 1991). However, fluorescence-labeled C5-ceramide clearly stained the cytoplasmic components in the rear part of the cell body and the uropod, in addition to the Golgi apparatus, in EL4.G8 cells (Fig. 6 A). In comparison to the MTOC or Golgi positioning, the intracellular arrangement of the front nucleus and the C5-ceramidelabeled rear cytoplasm was obviously stable (93.3% of cells showed this pattern). A similar staining pattern was observed in BW5147 cells bearing a uropod, although the MTOC and the Golgi apparatus in these cells were mostly detected in the rear cytoplasm (>95%; unpublished data). Thus, C5-ceramide was considered to be a useful probe for visualizing the posterior cytoplasm in EL4.G8 cells. Therefore, although they exhibit a slightly different feature of cell polarity from that observed in migrating lymphocytes, EL4.G8 cells show a unique asymmetry along the frontrear axis (Fig. 6 E).
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T567D ezrin augments cell migration
In general, the uropod is observed during lymphocyte migration (Sánchez-Madrid and del Pozo, 1999), suggesting that this structure might be important for efficient lymphocyte motility. EL4.G8 cells exhibited clear chemotactic migration toward a chemokine, stromal cellderived factor-1 (SDF-1), and this chemotaxis was prevented by pertussis toxin, which inactivates Gi-coupled chemokine receptor signaling, as well as by staurosporine and cytochalasin D (Fig. 7 A). Y-27632 also inhibited the chemotaxis, although withdrawal of the drug restored the activity (Fig. 7 B), indicating that the effects of Y-27632 are reversible. Therefore, ROCK activity is required for the proper chemotactic migration of EL4.G8 cells. Interestingly, among the transfectants of various ezrin mutants, only T567D ezrin transfectants showed higher chemotactic activity toward SDF-1, whereas other transfectants had activity comparable to that of the control cells (Fig. 7 C). We confirmed similar enhancement of chemotaxis in other transfectant clones for T567D ezrin. Together, these findings support the notion that ezrin phosphorylation and RhoROCK signaling regulate the efficiency of lymphocyte migration.
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Discussion |
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The formation of the polar cap seems to depend on both membrane anchorage and actin binding induced by CT phosphorylation of ERM proteins, but not on other interactive partners. It could be hypothesized that F-actin gathers multiple p-ERM molecules to "cross-link" them at one place underneath the plasma membrane. Under normal conditions, the p-ERM cap structure is likely to be reinforced by further construction of a kind of mature "uropod scaffold" that is mediated by other cellular components but sensitive to staurosporine. This seems reasonable, because even the ezrin mutants lacking actin-binding ability (such as NT and T567D-AB) are able to localize in the uropod, probably by the free NT domain, whereas staurosporine prevents this localization.
ERM proteins have been suggested to participate in Rho activation (Mackay et al., 1997; Sasaki and Takai, 1998; Tsukita and Yonemura, 1999). In vitro interaction of radixin and Dbl was previously reported (Takahashi et al., 1998). In this study, we showed that the protein complex containing phosphorylated ezrin exhibits Rho-GDP/GTP exchange activity, and that only the open-form ezrin can associate with Dbl through its NT domain. These findings suggest that accumulation of p-ERM is likely to augment the local activation of Rho beneath the uropod membrane. The overexpression of Dbl-AID reduced the ability of uropod formation in EL4.G8 cells, indicating that Dbl plays a substantial role in vivo. However, because various pathways can potentially induce Rho activation (Bishop and Hall, 2000; Ridley, 2001), it is also possible that Rho might be activated independently of ERM. The p-ERMDbl complex might be important for the maintenance of Rho activity restricted in the uropod membrane. There is a controversy concerning ROCK as a kinase for the CT Thr residues of ERM proteins, and this function has been suggested to depend on the cell type (Matsui et al., 1999; Yonemura et al., 2002). At least in EL4.G8 cells, ROCK is not responsible for ERM phosphorylation. In addition, though we treated EL4.G8 cells with a variety of chemical inhibitors for PKCs (BIM and Gö6976), PKA (KT5720), MEK (PD98059), p38MAPK (SB203580), or MLCK (ML-7), these drugs had no remarkable effect on the morphology of the cells. Thus, what maintains the p-ERM followed by the posterior RhoROCK signaling for uropod formation in EL4.G8 cells remains undetermined at this time.
The triggering of lymphocyte migration by various attractants involves complicated signaling (Ward et al., 1998; Hogg et al., 2003), although the overall picture of it remains incomplete. Environmental directional cues are thought to trigger the sequential signaling events upstream of the ERM phosphorylation, as well as the RhoROCK pathway, in unpolarized lymphocytes, and to induce the polarized morphology of lymphocytes that are able to migrate efficiently (Fig. 8). Our observations suggest that this process can be dissected into two steps: p-ERMmediated plasma membrane polarization and RhoROCK-mediated uropod protrusion (Fig. 8). In future studies, it will be important to identify any kinases that phosphorylate ERM CT Thr residues, to link the upstream signaling with p-ERM during lymphocyte migration.
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In addition, the uropod may function in effective tail retraction by concentrating the contraction machinery in the rear part of the cell. The RhoROCK pathway is known to be involved in a broad spectrum of cell contraction phenomena (Bishop and Hall, 2000; Ridley, 2001). The retraction of the trailing edge has been demonstrated to require this pathway in migrating leukocytes (Alblas et al., 2001; Worthylake et al., 2001; Smith et al., 2003), suggesting that these proteins are crucial for the maintenance of the posterior structure. Recently, it has been shown that the "backness" signal is generated by the activation of Rho by chemoattractant receptors in neutrophils (Meili and Firtel, 2003; Xu et al., 2003). In EL4.G8 cells, we show that Q63L RhoA, which is constitutively active independent of GEFs, is accumulated in the posterior cytoplasm. Because no association between ezrin and Rho could be detected (unpublished data), it can be postulated that there is some mechanism by which Rho-GTP is selectively anchored in the rear part of the cell, which is independent of p-ERM/Dbl function, or actively excluded from the leading edge.
Several reports have shown that Y-27632 induces a long tail that remains behind the cells because of blockage of the posterior retraction (Worthylake et al., 2001; Smith et al., 2003; Xu et al., 2003). However, the requirement of RhoROCK signaling for the uropod protrusion in the EL4.G8 model, as revealed by the ability of Y-27632 and dominant-negative Rho to completely abolish the uropod, seems to contradict the idea that the major function of this pathway is only for contraction. The uropod is usually observed as a membrane protrusion lifted off from, rather than attached to, the substratum in lymphocytes (Sánchez-Madrid and del Pozo, 1999). These facts indicate that the uropod, at least in lymphocytes, is not a simple tailing structure but rather is an active machinery for efficient contraction accompanied by somewhat of a protrusive effect, and thus is basically different from the trailing edges observed in adherent cells such as fibroblasts and epithelial cells.
A curious point is that the cytoplasmic polarity (i.e., the intracellular arrangement) of the nucleus, the MTOC, and the Golgi apparatus that is observed in migrating lymphocytes is, in general, in the opposite direction from that observed in many types of adherent cells with migrating morphology. In fibroblasts and astrocytes, the MTOC and the Golgi apparatus are positioned on the leading edge side, relative to the nucleus, during migration (Etienne-Manneville and Hall, 2002; Fukata et al., 2003). This suggests that the construction of the cell polarity in lymphocytes may follow different rules from those followed in adherent cells. Furthermore, although p-ERM is generally localized in an actin-rich compartment of the cell (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Bretscher et al., 2002), it is rather excluded from the actin-rich leading edge of migrating lymphocytes. It is likely that p-ERMspecific anchoring components exist in the posterior compartment in lymphocytes.
ERM proteins are involved in the integrity of the microvilli that constitute the apical compartment in epithelial cells (Mangeat et al., 1999; Tsukita and Yonemura, 1999; Gautreau et al., 2000; Bretscher et al., 2002). Specific localization of ERM proteins has also been reported at the nodes of Ranvier in Schwann cells (Melendez-Vasquez et al., 2001; Gatto et al., 2003). In Drosophila, the sole member of the ERM family, Dmoesin, has been demonstrated to be required for oocyte polarity (Polesello et al., 2002). However, the cellular compartmentalization in these examples is largely dependent on interactions with the neighboring cells or matrix. In highly motile immune cells, in contrast, the dependency of the cell morphology and polarity formation on the environmental structural support seems to be relatively weak. Hence, these cells might possess an intrinsic self-organizing system for plasma membrane polarization, using ERM proteins as the scaffold that connects selective transmembrane proteins to factors involved in cytoplasmic polarity.
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Materials and methods |
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Antibodies
The following antibodies or fluorescent probes were used in this study: phycoerythrin-antimouse CD43 and CD44 (BD Biosciences), FITCanti-CD43 (BD Biosciences), Alexafluor 546 phalloidin and Alexafluor 488 goat antirabbit IgG (Molecular Probes), antiß-tubulin (Sigma-Aldrich), FITCanti-GM130 (BD Biosciences), anti-GFP (CLONTECH Laboratories, Inc.), antimouse CD44 (KM201; IQ Products), antiezrin (Upstate Biotechnology), antip-ERM (Cell Signaling Technology), normal rabbit IgG anti-Dbl (Santa Cruz Biotechnology, Inc.), antiLFA-1 (FD441.8 hybridoma culture supernatant), and HRP-antirabbit IgG (Jackson ImmunoResearch Laboratories).
Immunofluorescence microscopy
For immunofluorescence staining, cells were fixed with 3% PFA in PBS and permeabilized with 0.2% Triton X-100/PBS or 0.5% saponin/PBS. To detect the intracellular localization of p-ERM, we performed a TCA fixation method, which inactivates phosphatases and maintains the level of p-ERM proteins during sample processing (Hayashi et al., 1999), with a slight modification. In brief, cells were fixed in suspension with 10% TCA solution for 15 min at room temperature, and were rinsed three times with PBS. For detection of the posterior cytoplasm, cells were labeled with BODIPY-FL or -TR C5-ceramide (Molecular Probes) for 30 min at 37°C, before fixation. After being blocked with 5% BSA-PBS, cells were stained with antibodies for 3060 min. The nucleus was visualized by staining with 0.1 µg/ml DAPI. Stained cells were mounted with Permafluor aqueous mounting medium (Immunotech) and examined with confocal laser scanning microscopes (model MRC-1024 [Bio-Rad Laboratories]; model TSC-SP2 [Leica]). Digital images obtained were processed by Adobe Photoshop software (Katakai et al., 2002).
Counting and measuring uropods and polar caps
We determined the number of cells bearing the uropod by counting cells in the digital images obtained with a phase-contrast microscope (model TND330; Nikon) equipped with a 40x objective and a measuring gauge. Images captured with a 3CCD camera (model C5810; Hamamatsu Photonics) were prepared with Adobe Photoshop software. The length of the uropod was determined by measuring the distance between the imaginary line of the rounded cell body and the tip of the protrusion. Polar caps (indicated by CD44 or GFP localization) formed in drug-treated or transfected cells were counted under a fluorescence microscope.
Plasmids
Expression vectors containing cDNAs encoding EGFP-tagged human WT ezrin, T567A ezrin, and T567D ezrin were provided by Monique Arpin (Institut Curie, Paris, France) (Gautreau et al., 2000; Coscoy et al., 2002). The NT fragment 1366 and CT fragment 466586 of ezrin were amplified from full-length ezrin cDNA by PCR, using the following oligonucleotide primers: EzrinNF-EcoRI, 5'-CGTGAATTCCCGAAAATGCCGAAACCAATC-3'; EzrinNIR-SalI, 5'-CGTCGTCGACTTCTCTGTTTCCAGCTGTTG-3'; EzrinCF-EcoRI, 5'-CGTGAATTCGTGATGACAGCA-CCCCCGCCC-3'; and EzrinCR-SalI, 5'-CGTGTCGACGACAGGCCCTCGAACTCGTCG-3'. Dbl-AID was amplified from EL4 cDNA using the following primer pair: N2-XhoI-F, 5'-CCCTCGAGTGGCATCGAATCACCACTATG-3' and N2-SalI-R, 5'-GGGGTCGACTGATCAGAATTCTTCTCTTGG-3'. The PCR products were subcloned into pEGFP-N1 (CLONTECH Laboratories, Inc.). To construct T567D-AB ezrin, we performed PCR using a plasmid vector for T567D ezrin-EGFP as a template and the following primer pair: SmaI-T567DF, 5'-GAAGGCCCAGGCCCGGGAGGAGAAGC-3' and AgeI-T567D-
ABR, 5'-CCGGACCGGTATGCGCTGCTTGGTGTTGCC-3'. The PCR products were subcloned into the T567D ezrin-EGFP plasmid. Expression vectors for EGFP-tagged WT, Q63L, and T19N Rho in a pcDNA3 background were a gift from Klaus Hahn (The Scripps Research Institute, La Jolla, CA) (Subauste et al., 2000).
Transfection
Cells (107 cells per 0.4 ml PBS) were transfected with 30 µg of plasmids in 4-mm-diam cuvettes by electroporation using a Gene Pulser (Bio-Rad Laboratories) at 380 V and 960 µF. After the clump of dead, lysed cells was removed, the cells were diluted 10-fold in medium (containing 10% FCS) and analyzed 24 h after electroporation. For stable transfection, cells electroporated with linearized plasmids were selected with 400 µg/ml G418 in 96-well plates. GFP-positive cells were further enriched by subcloning via limiting dilution, when necessary. Five stable clones for WT ezrin, T567A ezrin, and T567D ezrin, and two for NT ezrin, CT ezrin, and GFP-control vectors, were obtained.
Western blotting
Cells pretreated with or without 10% TCA were lysed in 5x SDS sample buffer. After the samples were boiled, equal amounts of total lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were soaked in a blocking solution (5% skim milk and 0.2% Tween 20-PBS) for 1 h, and then incubated with primary antibodies for 1 h. After being washed with Tween 20-PBS, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 h. Specific bands were visualized by an ECL method (ECL+; Amersham Biosciences).
Immunoprecipitation
Cells were lysed with RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, and protease inhibitors [1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2 µg/ml pepstatin A]) on a rotator for 1 h at 4°C. After insoluble materials were removed by centrifugation, the soluble supernatants were precleared with protein GSepharose 4 fast-flow (Amersham Biosciences). Samples were then immunoprecipitated with antibodies and 20 µl of protein G beads. Protein Gbound protein complexes were washed with lysis buffer and eluted by boiling in sample buffer for SDS-PAGE. The immunoprecipitated proteins were detected by Western blotting.
Detection of Rho guanine nucleotide exchange activity
To detect Rho-GEF activity associated with ezrin or p-ERM, we used an assay system combining immunoprecipitation, an in vitro exchange reaction, and pull-down detection of activated Rho. More than 3 x 108 cells were lysed with 2 ml RIPA buffer, and the protein complex was immunoprecipitated using an appropriate antibody. Alternatively, specific antibody (antip-ERM or anti-Dbl) was added to the lysate at the preclear step for the depletion of certain protein complexes. The in vitro exchange reaction was performed as described previously (Debant et al., 1996), with a slight modification. 0.5 µg of recombinant His6-RhoA (Cytoskeleton, Inc.) was preloaded with GDP incubating in 100 µl of loading buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1 mM DTT, and 1 mg/ml BSA) containing 10 µM GDP for 20 min at 25°C. After the incubation, the reaction was quenched with 100 µl of stop exchange buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM DTT), and then diluted with 1.5 ml of exchange buffer (50 mM Tris-HCl, pH 7.5, 200 µM GTPS, and 2 mM MgCl2). 500 µl of Rho-GDP solution was transferred to a tube containing the immunoprecipitated protein complex, and then incubated with gentle agitation on a rocker at 25°C for 20 min. Then, the reaction was quenched by adding 0.1 volume of stop buffer (50 mM Tris-HCl, pH 7.5, and 60 mM MgCl2). The amount of Rho-GTP in the reaction solution was measured by a pull-down method based on specific binding to Rhotekin-RBD followed by Western blotting using anti-Rho antibody (Rho activation assay biochem kit; BK306; Cyoskeleton, Inc.). The relative amount of active Rho compared with that in the control was calculated by measuring the band density of Rho and normalized to ezrin density.
Chemotaxis assay
The chemotaxis assay was performed using Transwell chambers (6.5-mm diam, 5-mm pore size, Costar). 200,000 cells suspended in 100 µl of medium were placed into the top chamber, and 600 µl of medium containing 10 ng/ml mouse SDF-1 (PeproTech) was added to the bottom well. Alternatively, cells were pretreated with 1 µg/ml pertussis toxin, 0.5 µM staurosporine, 10 µM Y-27632 (Calbiochem), or 10 µM cytochalasin D (Nacalai Tesque) in RPMI 1640 medium for 30 min at 37°C. After 4 h of chemotaxis, cells in the bottom well were collected and the cell number was counted using a FACScalibur flow cytometer (Becton Dickinson) with a constant time period (60 s) (Katakai et al., 2002).
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Acknowledgments |
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This work was supported in part by Grants-In-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The authors have no conflicting financial interests.
Submitted: 17 March 2004
Accepted: 14 September 2004
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References |
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