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Originally published In Press as doi:10.1074/jbc.M502639200 on June 23, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29653-29660, August 19, 2005
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Human T-lymphotropic Virus, Type 1, Tax Protein Triggers Microtubule Reorientation in the Virological Synapse*{boxs}

Mohamed Nejmeddine{ddagger}, Amanda L. Barnard{ddagger}, Yuetsu Tanaka§, Graham P. Taylor¶, and Charles R. M. Bangham{ddagger}||

From the {ddagger}Department of Immunology and the Department of Infectious Diseases, Wright-Fleming Institute, Imperial College, St Mary's Campus, Norfolk Place, W2 1PG, London, United Kingdom and the §Department of Immunology, Graduate School and Faculty of Medicine, University of Ryukyus, Uehara-cho 207, Nishihara-cho, Nakagami-gun, Okinawa 903-0215, Japan

Received for publication, March 10, 2005 , and in revised form, June 13, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We showed recently that the human T-lymphotropic virus, type 1 (HTLV-1), spreads directly from cell to cell via a virological synapse. The HTLV-1 virological synapse resembles the immunological synapse; each is a specialized contact between a lymphocyte and another cell that contains organized protein microdomains, and each involves repolarization of the T-cell microtubule cytoskeleton. However, formation of the virological synapse is not triggered by T-cell receptor-mediated antigen recognition. On the basis of our previous data, we postulated that formation of the viral synapse was triggered by a conjunction of two signals, one from HTLV-1 infection of the T-cell and one from cell-cell contact. We have recently identified ICAM-1 engagement as a cell-contact signal that causes the microtubule polarization associated with the virological synapse. Here we used confocal microscopy of T-lymphocytes naturally infected with HTLV-1 or transfected with individual HTLV-1 genes to investigate the role of the viral transcriptional transactivator protein Tax. Polarization of the microtubules was induced by cell-cell contact or by cross-linking T-cell surface molecules with monoclonal antibodies adsorbed to latex beads. We show that Tax, which is mainly found in the nucleus, is also present at two specific extranuclear sites as follows: around the microtubule organizing center in association with the cis-Golgi and in the cell-cell contact region. We show that expression of Tax provides an intracellular signal that synergizes with ICAM-1 engagement to cause the T-cell microtubule polarization observed at the virological synapse.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human T-lymphotropic virus, type 1 (HTLV-1),1 is the etiological agent of adult T-cell leukemia/lymphoma and is also associated with several inflammatory disorders, including HTLV-1-associated myelopathy/tropical spastic paraparesis. Naturally infected lymphocytes produce virtually no cell-free infectious HTLV-1 in vivo, and cell contact is required for efficient transmission of HTLV-1 between cells and between individuals. It has been shown recently that when an HTLV-1-infected T-cell meets an uninfected T-cell, the microtubule-organizing center (MTOC) of the infected cell becomes polarized toward the uninfected cell. Complexes of HTLV-1 core (Gag) and the RNA genome of the virus accumulate at the point of cell contact and are then transferred to the uninfected cell (1).

The establishment and maintenance of cell polarity is an essential feature of all eukaryotic cells, both during development and in the adult. In migrating adherent cells such as fibroblasts and endothelial cells, the MTOC reorients toward the leading edge of a wounded monolayer (2, 3). Migrating macrophages also localize their MTOC in a position between the nucleus and the leading edge of the cell. A similar reorientation of the MTOC occurs in CD4+ T-cells directed toward an antigen-presenting cell that carries the cognate antigen peptide (4) and in natural killer cells and cytotoxic T lymphocytes when they interact with their respective target cells (5, 6). The MTOC of the responding lymphocyte reorients toward the cell-cell contact region, where these interactions occur. This reorientation occurs rapidly, and the reorienting MTOC may be accompanied by other components or organelles, such as the Golgi apparatus (5).

Genetic analysis of the budding yeast Saccharomyces cerevisiae has revealed that the small GTPase Cdc42 is an essential regulator of cell polarity (7). Cdc42 has also been shown to regulate polarity in mammalian cells. For example, inhibition of Cdc42 prevents the following: (i) the reorientation of the MTOC in T-cells in response to an antigen-presenting cell (8); (ii) the directional movement of macrophages toward a chemotactic signal (9); (iii) the directional movement and the reorientation of the Golgi of fibroblasts in an in vitro wound assay (10); and (iv) polarized basolateral secretion/endocytosis in the epithelial cell line (Madin-Darby canine kidney cells) (11).

By having established the effect of cell contact on the orientation of the HTLV-1-infected MTOC of the T-cell and the distribution of the HTLV-1 Gag protein (1), we wished to study the intracellular distribution of the HTLV-1 transcriptional activator protein (Tax) and its possible role in the formation of the virological synapse. Tax is among the first HTLV-1 proteins to be translated in ex vivo cultured cells (12). It has been well established that the HTLV-1 Tax protein plays a critical role in cellular transformation (13, 14). Tax not only activates the expression of HTLV-1 genes through the viral long terminal repeat, but also stimulates the transcription of a number of cellular genes through cellular signaling pathways of NF-{kappa}B, CREB, serum-response factor, and AP-1. Tax does not bind to promoter or enhancer sequences by itself but by interacting with a number of transcription factor families, including NF-{kappa}B, cyclic AMP-response element-binding protein/activated transcription factor, and the CREB-binding protein CBP/p300 to stimulate the transcription from the respective promoters.

Tax protein has been shown to be a major target of cytotoxic T lymphocytes in vivo (15-17). Depletion of CD8+ T-lymphocytes from the peripheral blood mononuclear cells of HTLV-1-infected individuals in vitro promoted Tax expression in the CD4+ subpopulation, suggesting that CD8+ cytotoxic T lymphocytes suppress Tax expression in vivo (12).

The aim of the present study was to investigate the effect of cell-cell contact on the intracellular distribution of Tax protein, and to test the hypothesis that Tax protein is sufficient to account for the strong polarization of the MTOC associated with the HTLV-1 virological synapse.

We report that Tax protein is present not only in the nucleus of infected cells (18, 19) but also in two extranuclear sites as follows: around the MTOC in close association with the cis-Golgi compartments and in the cell-cell contact region. Reorientation of the MTOC is always accompanied by Tax protein reorientation in the same direction. This MTOC/Tax polarization is dependent on the integrity of the cytoskeleton and on the function of the Rac family and Cdc42 small GTPases. Tax protein expression is sufficient to induce the polarization of the MTOC toward the target cell. The results suggest a previously unidentified role for Tax protein in promoting cell to cell spread of HTLV-1.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—To detect HTLV-1 proteins in infected cells, we used the mouse IgG3 monoclonal antibody (mAb) directed against HTLV-1 Tax protein (Lt-4) (20) and the mouse IgG2b mAb directed against HTLV-1 Gag p19 (Gin7) (21). The microtubule network was stained with the anti-{beta}-tubulin (CY 3-conjugated) mAb TUB2.1 obtained from Sigma. The Golgi apparatus was detected with the mouse IgG1 mAb anti-GM130 (BD Transduction Laboratories), anti-giantin polyclonal antibody (CRP Inc.; Cambridge Bioscience Ltd., Cambridge, UK), or with anti-ERGIC53 mAb (22) a gift from Hans-Peter Hauri (Basel, Switzerland). Alexa fluorophores (488, 564, and 645 nm) conjugated to isotype-specific antibodies were purchased from Molecular Probes, Eugene, OR. Antibodies used to coat the latex beads were as follows: anti-CD54 (ICAM-1), clone HA58 (Research Diagnostics Inc.) and anti-CD11a, clone 38 (mouse IgG2a). The nucleus was stained with 4',6-diamidino-2-phenyindole, dilactate (DAPI; Sigma).

To gain further insight into the biochemical pathways that control microtubule polarization, we used a number of agents to treat infected cells. Nocodazole, latrunculin B, brefeldin A, and lithium chloride (an inhibitor of GSK-3, which inhibits MTOC polarization) were purchased from Sigma and were used at a final concentration of 10, 5, and 15 µM and 20 mM, respectively. Toxin B (Clostridium difficile) is a high molecular weight glucosyltransferase that inhibits small GTPases such as Rho, Rac, and Cdc42 by glycosylation of a threonine residue; exoenzyme C3 (Clostridium botulinum) inhibits Rho GTPases by catalyzing the ribosylation of Asn-14; and GF109203X, a cell-permeable inhibitor of protein kinase C (PKC), shows high selectivity for PKC{alpha},-{beta}I, -{beta}II, -{gamma},-{delta}, and -{epsilon} isozymes. These drugs were purchased from Calbiochem and were used at a final concentration of 0.4 pM, 0.2 µM, and 20 µM, respectively (23).

Naturally Infected T Cells and Cell Lines—PBMCs were obtained from HTLV-1-seropositive and -associated myelopathy/tropical spastic paraparesis patients with a high proviral load attending the National Centre for Human Retrovirology at St. Mary's Hospital, London, UK. All patients gave informed consent. The HTLV-1-immortalized cell line MS-9 was a kind gift from Dr. David Derse, NCI, National Institutes of Health. MS-9 cells were derived by co-culture of phorbol 12-myristate 13-acetate-activated human PBMCs with DBS-FRhL (clone B5) cells that were infected with the HTLV-1 molecular clone, pHTLV-X1MT (24). MS-9 cells were cultured in RPMI 1640 (Sigma) supplemented with 2 mM glutamine (Invitrogen), 100 IU/ml penicillin (Invitrogen), 100 IU/ml streptomycin (Invitrogen), 20% heat-inactivated fetal calf serum (PAA Laboratories Ltd., Somerset, UK), and 100 units/ml recombinant interleukin 2 (Sigma). Jurkat cells (clone E6.1) were obtained from ATCC, Middlesex, UK. Jurkat E6.1 is a clone of the Jurkat-FHCRC cell line, a derivative of the Jurkat cell line (25).

Isolation of CD4+ T Cells—PBMCs were isolated by density gradient centrifugation on Histopaque-1077 (Sigma), washed twice with phosphate-buffered saline (PBS) and washed once in PBS, 10% fetal calf serum. CD4+ cells were negatively selected from the PBMCs by using the CD4+ T-cell isolation kit from Miltenyi Biotech, Surrey, UK, following the manufacturer's instructions. This procedure yielded CD4+ cells at a purity of greater than 95%, ascertained by flow cytometry (data not shown). Before use, the isolated CD4+ T-cells were cultured overnight in 10-cm diameter tissue culture dishes (106 cells/ml) to allow spontaneous expression of HTLV-1 proteins (12). During this incubation, the cells were widely dispersed to minimize cell-cell contact. T-cells were cultured in RPMI 1640 medium (Sigma) supplemented with 2 mM glutamine (Invitrogen), 100 IU/ml penicillin (Invitrogen), 100 IU/ml streptomycin (Invitrogen), and 20% heat-inactivated fetal calf serum (PAA Laboratories Ltd., Somerset, UK).

Immobilization of Antibodies on Latex Beads—For each antibody, 80 x 106 surfactant-free sulfate white latex 5-µm beads (Interfacial Dynamics Corp., Portland, OR) were washed twice in 10 ml of 0.025 M MES buffer, pH 6.1 (Sigma). The beads were centrifuged for 20 min at 3000 x g and resuspended in 1 ml of the MES buffer. One hundred µg of the respective antibody was added in a 15-ml centrifuge tube and incubated overnight at room temperature on a roller, with constant mixing. The beads were then washed twice in 10 ml of PBS and resuspended in 1 ml of PBS, 1% bovine serum albumin. The antibody-coated beads were stored at 4 °C. Flow cytometric analysis was used to verify that the beads were labeled with antibody (results not shown).

Conjugate Formation and Analysis of MTOC Polarization—To form cell-cell conjugates (or cell-bead conjugates), 5 x 104 cells were placed in each well (6 mm diameter) of a 10-well glass slide (VWR Scientific, Magma Park, Lutterworth, UK). The slides were treated for 5 min at room temperature with 50 µl/well of 100 µg/ml poly-L-lysine (Sigma) and then left to dry before use. The cells were left to adhere for 30 min at 37 °C, and then 105 cells or 105 antibody-coated beads were added to the adherent cells at a ratio of 2:1 (2 cells or beads added to 1 adherent cell). The conjugate formation was left to occur for 1 h at 37 °C. The cells were then fixed with 4% paraformaldehyde at room temperature, and immunofluorescence staining was performed as described below.

Cell-bead conjugates were first identified by differential interference contrast using a Laborlux 12 Leitz fluorescent microscope equipped with a water-based objective (x50/1.0). Between 50 and 100 events were counted per condition per experiment. T-cells were scored as conjugates where the infected cell was not in contact with another cell and where there was only one point of contact between the cell and a bead. We considered that the MTOC and Tax protein were polarized when their orientation to the beads was located within a quarter of the cell circumference.

Plasmids and Transfection—For transient transfections, Jurkat cells (clone E6.1) were transfected with 5 µg of plasmid DNA using a NucleofectorTM device (Amaxa Inc., Gaithersburg, MD) according to the manufacturer's optimized protocol for Jurkat cells (program S-18).

Jurkat cells were transfected with the full-length tax gene in the pJFE-tax plasmid (26), in which the gene was under the control of the SR{alpha} promoter, or with the Tax mutant M47. This mutant was obtained by the substitution of two amino acids near the COOH-terminal end (L319R, L320S). Tax (M47) is a nonfunctional mutant, it has been demonstrated previously to abrogate selectively the ability of Tax to activate transcription through CREB/activated transcription factor pathway (27) (28). The amino acids substitution was introduced into Tax-GFP template using the QuickChange® site-directed mutagenesis kit (Stratagene, La Jolla, California), according to the manufacturer instructions, and was verified by DNA sequencing (data not shown).

ICAM-1 Blocking Peptide cLAB.L—Jurkat cells transfected with Tax protein (2.5 x 106/ml) were washed in serum-free RPMI and were incubated with 200 µM of cLAB.L peptide at 37 °C for 30 min (29). The cells were washed twice in RPMI serum-free medium to eliminate the excess peptide; then unblocked Jurkat cells were added, and conjugates were allowed to form for 30 min at 37 °C.

Indirect Immunofluorescence Staining for Confocal Microscopy— HTLV-1-infected cells were fixed with 4% paraformaldehyde for 30 min at room temperature. For intracellular immunofluorescene staining, cells were permeabilized with 1% Triton X-100 (Sigma) in PBS for 10 min at room temperature and then were incubated with 5 µg/ml of the primary antibodies (anti-Tax, anti-Gag p19, or anti-GM130) for 1 h at 37 °C, followed by incubation with 1 µg of the respective secondary antibody Alexa-conjugated goat anti-mouse immunoglobulin G (IgG3, IgG2a, or IgG1) for 30 min at 37 °C. The antibodies were applied sequentially, and the cells were washed three times between each step with PBS containing 50 mM NH4Cl. The nucleus of the cell was stained with 5 µg/ml DAPI for 5 min at room temperature and then washed twice with PBS containing 50 mM NH4Cl. After the last wash, cells were mounted with Glycergel (Dako Corp., Carpinteria, CA).

Confocal microscopy analysis was carried out using the Zeiss Pascal LSM confocal imaging system (Zeiss, Heidelberg, Germany), equipped with a 63x/1.4 oil objective.

A krypton-argon mixed gas laser was used to generate three bands: 488 nm for Alexa-488 (fluorescein isothiocyanate), 568 nm for Alexa-568 (TRITC or CY3), or 645 nm for Alexa-645 (CY5); and a UV laser was used to detect DAPI staining. Multichannel acquisition was used to avoid cross-talk between the respective channels. The signal was treated with line averaging to integrate the signal collected over four lines in order to reduce noise. The pinhole was adjusted to allow a field depth of about 0.5 µm, corresponding to the increment between two adjacent sections. Image processing was performed with the on-line LSM 5 image browser (Zeiss, Heidelberg, Germany). Numeric images were transferred to a computer equipped with an image analysis station (Scion image and Photoshop 7.0).

Statistical Analysis—The frequency of MTOC polarization in HTLV-1-infected and uninfected cells was compared by using the odds ratio (OR). To test whether a given OR differed significantly from 1.0, and to compare two different odds ratios, we applied normal theory to the distribution of ln(OR), the natural logarithm of the OR (30). To calculate the summary odds ratio for a given effect over a number of experiments, we used the inverse variance method of weighting individual values of ln(OR) (30). To calculate the statistical significance of a given effect over different experiments, we used Fisher's {chi}2 method of combining probabilities as shown in Equation 1,

(Eq .1)
where pi is the significance level obtained from the ith experiment, and k is the number of experiments. -2{Sigma}ln(pi) is distributed as a {chi}2 variant with 2k degrees of freedom (31).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In Naturally Infected T Cells, a Substantial Fraction of HTLV-1 Tax Protein Was Extranuclear and Colocalized with the MTOC—In isolated T-cells from HTLV-1-infected subjects, Tax protein was mostly expressed in the nucleus of CD4+-infected T-cells (Fig. 1A). However, a significant fraction of Tax protein was present outside the nucleus (Fig. 1). A rotatable three-dimensional view of Fig. 1, A-C, showed the extranuclear fraction of Tax protein (supplemental data Fig. S1). The superposition (in yellow) of Tax protein staining in green (Fig. 1A) and microtubule network staining in red (Fig. 1B) showed that the extranuclear fraction of Tax protein lay adjacent to the MTOC in infected T-cells (Fig. 1C). Digital zooming on single cells confirmed the juxtaposition of extranuclear HTLV-1 Tax protein and the MTOC (Fig. 1D). This result was confirmed by staining the nucleus of HTLV-1-infected MS9 cells (continuous cell line) with DAPI (supplemental data Fig. S2). Fig. S2 demonstrated the presence of Tax protein outside the nucleus and around the MTOC.

We then investigated the distribution of HTLV-1 Tax protein in comparison to Gag p19 complexes in CD4+ T-cell conjugates. The conjugates were formed spontaneously between autologous PBMCs from HTLV-1-positive individuals, as described previously (1). During the cell-cell contact both Gag p19 complexes and Tax protein were found at the cell-cell junction and around the MTOC (Fig. 2). In both cases the MTOC was oriented toward the uninfected T-cell (Fig. 2, B-D). We have never seen Tax protein be transmitted to the uninfected target cell, in contrast to the regular transmission of Gag p19 complexes (1).2



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  		FIG. 1.
Intracellular distribution of HTLV-1 Tax protein. Naturally infected CD4+ T-cells purified from the PBMCs of seropositive donors were stained with anti-Tax mAb Lt-4 (green) and anti-{beta}-tubulin mAb CY3 conjugated (red). HTLV-1 Tax protein (A), anti-{beta}-tubulin (B), superposition of A and B (C). High magnification of HTLV-1-infected cell stained with anti-Tax protein and with anti-{beta}-tubulin (D). Each arrow indicates an MTOC; C and D, the MTOC (red) is adjacent to Tax protein (Green). Scale bar, 10 µm.

 



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  		FIG. 2.
Intracellular localization of Tax protein and Gag p19 complexes in HTLV-1-infected T-cell conjugates. Naturally infected T-cells (CD4+) purified from the PBMCs of HTLV-1-seropositive donors. Each panel shows the superposition of staining with anti-{beta}-tubulin mAb (red) and anti-Gag p19 mAb or anti-Tax protein mAb (green). A shows unpolarized Gag protein in isolated T-cell. B-D show polarization of Gag protein (B and C) or Tax protein (D) in autologous T-cell conjugates. D, the polarized Tax protein is seen as a green crescent along the cell-cell junction; most of the Tax staining remains in the nucleus. Scale bar, 10 µm.

 
To test whether the distribution of Tax protein in infected cells is modified in the absence of virus replication, Jurkat cells were transiently transfected with pJFE-Tax DNA plasmid. The expression of Tax protein was found to be maximal 24 h after the transfection; at this time Jurkat cells were fixed and permeabilized and then submitted to immunofluorescence staining with the anti-Tax protein monoclonal antibody (Lt-4) and with anti-{beta}-tubulin. Tax protein was expressed in 20-30% of the transfected cell population as detected by the specific monoclonal antibody (Lt-4). The results (Fig. 3) showed that in both transfected Jurkat cells (Fig. 3C) and the HTLV-1-infected cell line (MS9) (Fig. 3A), Tax protein followed a pattern similar to that observed in CD4+ T-cells naturally infected with HTLV-1 (Figs. 1 and 2). Indeed, Tax protein within the cytoplasm was distributed around the MTOC and in the cell-cell contact region of spontaneous cell-cell conjugates.



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  		FIG. 3.
Intracellular localization of Tax protein in HTLV-1-infected cell line (MS9) and in transfected cell conjugates. MS9 cells were stained with anti-Tax mAb Lt-4 (green) and with anti-{beta}-tubulin mAb (red) (A). Jurkat cells were transiently transfected with (pJFE-Tax) and then stained with anti-Tax mAb Lt-4 (E) and anti-{beta}-tubulin mAb (F). C corresponds to the merged images of E and F. B shows transmission light image of the MS9 cell conjugate in A; D shows transmission light image of the Tax-transfected conjugate in C. Scale bar, 10 µm.

 
Tax Protein Subcellular Localization Coincided with Golgi Stacks—We showed above that there is a strong spatial association between the MTOC and Tax protein within the cytoplasm in both infected and transfected cells. Furthermore, the Tax protein pattern and localization revealed by immunofluorescence staining suggested that Tax protein is associated with large vesicular structures similar to the Golgi compartments present in the proximity of the MTOC. To test whether Tax indeed colocalizes with a Golgi compartment, we performed a triple immunofluorescence staining of the Tax protein, the microtubules, and the Golgi compartments in naturally infected CD4+ T-cells, using the anti-Tax mAb (Lt-4), the anti-{beta}-tubulin mAb, and several Golgi markers, respectively (Fig. 4). The Golgi was stained with antibodies directed against the Golgi matrix protein 130 (GM130), or the Giantin protein, or the ERGIC53 protein, which is an endoplasmic reticulum intermediate Golgi marker. To analyze the colocalization between Tax protein and the Golgi stacks, we compared the images corresponding to the superposition of Tax and Golgi staining obtained in the same confocal sections. The results are shown in Fig. 4. We found a partial colocalization between HTLV-1 Tax protein and the Golgi complex (Fig. 4, top panel). The anti-Giantin and the anti-ERGIC53 staining were also found to coincide with Tax protein staining (data not shown). These antibodies have the common feature of recognizing the cis-Golgi compartments. The juxtaposition between HTLV-1 Tax protein and the Golgi was observed both in naturally infected cells (Fig. 4, top panel) and in the HTLV-1-infected cell line MS9 (data not shown).

The treatment of naturally infected cells with nocodazole (Fig. 4, middle row) or brefeldin A (Fig. 4, bottom row) resulted in fragmentation of the Golgi complex to multivesicular structures dispersed within the cytoplasm. Despite the dispersal of the Golgi complex, Tax protein remained adjacent to the fragmented Golgi stacks. The same observation was made in the presence of brefeldin A. These results confirmed that Tax protein was strongly associated with the Golgi complex.

MTOC and Tax Protein Were Both Polarized toward the Cell-Cell Contact Region—As described previously (1), we observed that the MTOC in HTLV-1-infected cells was oriented preferentially toward the uninfected cell. This MTOC polarization was always accompanied by Tax protein polarization in the same direction.

MTOC polarization can be artificially induced by cross-linking specific T-cell surface proteins, such as T-cell receptor, LFA-1, ICAM-1, with specific antibodies (32-34). To quantify the frequency of copolarization of the MTOC and Tax protein in infected T-cells, we used latex beads coated with an anti-ICAM-1 mAb to induce a high frequency of MTOC polarization.

The results (Fig. 5, left-hand columns) showed that the cross-linking of ICAM-1 with specific antibody was sufficient to induce MTOC polarization to the cell-bead contact region in nearly 100% of cells, whereas treatment with a control anti-IgG1 antibody had no such effect. Furthermore, Tax protein was always oriented in the same direction, in agreement with the observation made above concerning the association of the MTOC and Tax protein in HTLV-1-infected cells.

MTOC/Tax Polarization Was Abolished by Agents That Depolymerize Microtubules or Microfilaments—If Tax protein is indeed associated with the MTOC, then the depolymerization of the cytoskeleton would be expected to alter the orientation of Tax protein toward the latex beads coated with anti-ICAM-1 mAb. To test this prediction, we investigated the effect of agents that depolymerize microtubules or microfilaments on the MTOC/Tax protein polarization. T-cells naturally infected with HTLV-1 (or MS9 cells) were treated with nocodazole (10 µM, 1 h at 37°C) or with latrunculin B (5 µM, 1 h at 37 °C). The latex beads coated with anti-ICAM-1 mAb were then added to the cells and incubated for 1 h at 37°C. The results (Fig. 5) show that the treatment with either nocodazole or latrunculin B efficiently abolished the orientation of both MTOC and Tax protein to the cell-bead contact region. Even so, the MTOC and Tax protein remained strongly associated in the cytoplasm of the HTLV-1-infected T-cells. These results demonstrate that the integrity of the microtubule and the microfilaments was required for MTOC/Tax orientation to the cell-bead contact region and that Tax protein was always associated with the MTOC, regardless of the integrity of microtubules or microfilaments.

MTOC/Tax Polarization Was Abolished by the Inactivation of Small GTPases—MTOC polarization is known to be dependent on the activity of certain small GTPase proteins (23, 35). To investigate the involvement of these molecules in the orientation of the MTOC and Tax to the cell-cell contact region, we incubated HTLV-1-infected T-cells with several drugs known to inactivate the small GTPase proteins, or drugs that block the PKC pathway. By using the same experimental conditions as those described previously in the absence of the drugs, naturally infected CD4+ T-cells (or the MS9 cell line) were incubated with toxin B (1 pg/ml, 3 h) to inactivate both Rho, Rac, and Cdc42 small GTPases or with exoenzyme C3 (toxin C3, 5 µg/ml, overnight) to selectively inactivate Rho GTPase. We also used the drug GF109203X (20 µM, 1 h) to selectively block the PKC pathway and LiCl as a positive control for the inhibition of MTOC polarization.



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  		FIG. 4.
Colocalization of Tax protein with the Golgi stacks in naturally infected T-cells. Naturally infected T-cells were stained with anti-Tax mAb Lt-4 (green) and with anti-{beta}-tubulin (red); the cis-Golgi compartment was stained with anti-GM130 (blue); the merged image corresponds to the superposition of Tax, {beta}-tubulin, and Golgi. The center panel shows HTLV-1-infected cells treated with nocodazole. The lower panel shows HTLV-1-infected cells treated with brefeldin A. Arrows indicate the association between Tax protein and cis-Golgi stacks. Scale bar, 10 µm.

 
The results (Fig. 5) show that the toxin B abolished the orientation of both the MTOC and Tax protein to the cell-bead contact region. In contrast, treatment with either toxin C3 or GF109203X (Fig. 5) did not abolish the orientation of the MTOC or Tax to the bead contact region. Regardless of the drug treatment, the MTOC and Tax protein remained associated in the HTLV-1-infected T-cells. The distinct effects of toxin B and toxin C3 showed that MTOC/Tax polarization was reduced only by the inactivation of Rac or Cdc42 small GTPases but not by inactivation of Rho GTPases or enzymes in the PKC pathway.

Tax Protein Expression Is Sufficient to Facilitate MTOC Polarization Induced by Cell-Cell Contact or ICAM-1 Cross-linking—In order to investigate the involvement of HTLV-1 Tax protein in the MTOC polarization, we transiently transfected Jurkat cells with the pJFE-Tax expression plasmid. Cell-cell conjugates were allowed to form 24 h after the transfection; the cells were then fixed and stained for Tax protein and microtubules. The effect of Tax on MTOC polarization was quantified by counting the frequency of MTOC orientation in cells expressing Tax protein, Tax mutant protein (M47), or GFP alone toward the region in contact with untransfected cells (Table I). The results (Fig. 6) show that in nearly 80% of Jurkat cells expressing Tax protein the MTOC was oriented to the cell contact junction. From five independent experiments the composite OR of MTOC polarization in Tax+ cells compared with Tax- cells was 11.9 ({chi}2 = 198; 8 degrees of freedom; p << 0.001), whereas the Tax mutant (M47) and the GFP vector alone had no effect (Tax M47 composite OR = 0.83; p > 0.05) (GFP alone composite OR = 0.7; p > 0.05). Pretreatment of cells with the cyclic peptide cLAB.L, which blocks the interaction of ICAM-1 with LFA-1 (29), abolished the MTOC polarization associated with Tax protein (Table I).


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  		TABLE I
Effect of tax protein expression on MTOC polarization in Jurkat cells In each representative experiment the values show the number of events (MTOC polarization to the cell-cell junction) observed.

 



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  		FIG. 5.
Effect of anti-cytoskeleton drugs or enzyme inhibitors on MTOC and Tax polarization. MTOC polarization was induced by cross-linking of ICAM-1 on the surface of HTLV-1-infected T-cells with latex beads coated with anti-ICAM-1 antibody. The results represent the percentage of HTLV-1-infected CD4+ T-cells whose MTOC and Tax protein were polarized toward the cell-bead contact region. Between 50 and 100 events were counted per condition per experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tax protein contains a leucine-rich nuclear export signal (36) and also a nuclear localization sequence near its amino terminus (amino acids 2-58) (37). Previous studies have indicated that Tax is able to shuttle between the nucleus and the cytoplasm (38). Cytoplasmic localization of Tax protein could result from lack of nuclear localization sequence recognition and nuclear import shortly after protein synthesis or exit from the nucleus via a cellular nuclear export system. This is not an unusual functional feature of proteins involved in transcriptional regulation because several reports have demonstrated that differential localization of such proteins can function to either inhibit or induce a cellular process (39, 40). Cytoplasmic Tax protein activates NF-{kappa}B by binding directly to NF-{kappa}B family members (including NF-{kappa}B p50, p65, c-Rel, and NF-{kappa}B2) and by destabilizing the NF-{kappa}B inhibitor I{kappa}B (41). Tax protein interaction with the human mitotic checkpoint protein MAD1 in the cytoplasm of HTLV-1-infected cells, the resulting loss of cell checkpoint control, and the appearance of multinucleated giant cells may be the result of extranuclear actions of Tax protein (42). Meertens et al. (43) recently reported that Tax2 protein, from the closely related virus HTLV-2, is located predominantly in the cytoplasm, although its precise subcellular localization was not described. In HTLV-1-infected cells, Tax protein was predominantly nuclear, but inhibition of proteasome function caused an increase in the level of Tax protein in the cytoplasm. However, no data were presented on the localization of Tax within the cytoplasm or on the mechanisms that control this localization. It has been recently shown that although it was ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitin-proteasome pathway, suggesting that Tax conjugation to ubiquitin mediates a non-proteolytic function (44).



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  		FIG. 6.
Effect of Tax protein expression on MTOC polarization in Jurkat cells. The results represent the percentage of Jurkat cells expressing Tax or GFP proteins whose MTOC was polarized toward the cell-cell contact region. Between 50 and 100 events were counted per condition per experiment. N indicates the repetition of the experiments. wt, wild type.

 
Peloponese et al. (45) suggested that that ubiquitination does not target Tax for degradation by the proteasome. Rather, ubiquitin addition modified Tax in a proteasome-independent manner from an active to a less-active transcriptional form (45).

In this report, we show that although most Tax protein was present in the nucleus it was also found at two specific extranuclear sites. One extranuclear fraction of Tax protein was closely associated with the MTOC, and its distribution suggested that it was closely associated with the cis-Golgi compartment. In addition, HTLV-1 Tax protein was present in the cell-cell junction in conjugates formed between HTLV-1-infected and uninfected T-cells. The presence of Tax in the cell-cell contact region was always accompanied by the orientation of the MTOC of the infected cell toward the other cell in the conjugate. Previous work (1) showed the orientation of the MTOC and HTLV-1 Gag protein in infected T-cells toward the target cells. However, although complexes containing Gag protein and the HTLV-1 genome were frequently transferred across the virological synapse from one cell to the other, we have never observed intercellular transfer of Tax protein, even in conjugates in which large quantities of Tax protein accumulated at the cell-cell junction.

The copolarization of Tax protein and the MTOC may be explained by the apparent association of Tax protein with the cis-Golgi. Indeed, the Golgi stacks are usually arranged as an interconnected network in the region around the MTOC. After drug-induced disruption of microtubules, the Golgi stacks are disconnected from each other, partly broken up, dispersed in the cytoplasm, and redistributed to endoplasmic reticulum exit sites (reviewed in Ref. 46).

Depolymerization of the microtubules or the microfilaments abolished the polarization of both Tax and the MTOC to the cell contact region. Nevertheless, Tax protein remained closely associated with the MTOC in the cytoplasm of infected T-cells. It has been established that Tax protein interacts with several elements of the cytoskeleton. The {alpha}-internexin, a neuronal intermediate filament protein, was first identified in vitro by using the two-hybrid system (47). Tax protein also interacts with cytokeratin intermediate filaments (48). Recent proteomic studies suggest that Tax protein interacts with cytoskeleton proteins families, such as gelsolin and actin, and with certain proteins that influence cytoskeleton dynamics, such as annexin, myosin light chain kinases, and myotubularin-related protein. Tax protein also interacts with several small GTPases including Cdc42, RhoA, and Rac1 (49). These proteins are known for their regulatory role in actin dynamics leading to cellular transformation (50-52). However, many lines of evidence point to a regulatory role of these small GTPases in microtubule dynamics as well. MTOC polarization is blocked by either the dominant negative form or the constitutively active form of Cdc42 (8). These observations have been extended in other studies in different cell models, such as migrating astrocytes, demonstrating that activated Cdc42 acts through a Par6-atypical PKC complex. Cdc42 regulates cell polarity by spatial regulation of GSK-3{beta} (23, 35). It has also been reported that MTOC polarization depends on Cdc42, dynein, and dynactin (53). Rac1 has been reported to mediate microtubule stabilization through phosphorylation and the inhibition of the microtubule-destabilizing protein stathmin by PAK1 (54). In our experiments, the inactivation of small GTPases, in the Rac and Cdc42 family, resulted in the abolition of polarization toward the cell contact junction. This result was in accordance with the previous studies.

Tax protein expressed in Jurkat cells showed a distribution similar to that observed in naturally infected T-cells. This result demonstrates that Tax protein distribution in HTLV-1-infected T-cells is not necessarily dependent on HTLV-1 replication or on the expression of other HTLV-1 proteins. The presence of Tax protein in juxtaposition to the cis-Golgi compartments is not because of functional maturation of the protein because the Tax protein has no known signal peptide and is not glycosylated. These results, taken together, suggest that the Tax protein is associated with a cellular protein that targets the MTOC-Golgi region and subsequently the cell-cell junction.

As described previously (1), we observed frequent intercellular transfer of Gag complexes. However, we never observed transmission of the Tax protein to an uninfected cell. HTLV-1 Tax has been shown to bind certain proteins present in the cytoplasm; these interactions may play a part in HTLV-1-induced transformation of the cell (42). Our observation that Tax is present near the MTOC and at the cell-cell contact region and the regulation of the MTOC polarization by HTLV-1 Tax protein expression suggest that Tax protein is actively involved in the cytoskeletal reorientation and tight cellular adhesion that characterize the HTLV-1 virological synapse (1).

Finally, we showed that Tax protein expression was sufficient to explain the high frequency of the MTOC polarization toward the cell contact region that is observed at the HTLV-1 virological synapse. Blocking the interaction between ICAM-1 and LFA-1 abolished this preferential polarization. We have shown recently that engagement of ICAM-1 on the surface of an HTLV-1-infected cell is sufficient to explain the observed MTOC polarization in the HTLV-1 virological synapse (55). We conclude that Tax protein expression reduces the threshold of T-cell MTOC polarization induced by ICAM-1 cross-linking. Because cross-linking of ICAM-1 has itself been shown to increase HTLV-1 protein expression (56), such a positive feedback pathway could provide an efficient mechanism to promote the cell-cell spread of HTLV-1.

The mechanism remains to be identified by which Tax protein sensitizes a T-cell to polarization of the microtubule cytoskeleton induced by engagement of ICAM-1. ICAM-1 engagement activates both Rho/Rac/Cdc42 and Ras small GTPases (57). A role for RhoA is excluded here by the lack of effect of toxin C3 on cytoskeletal polarization. However, activation of Rac/Cdc42 culminates in the activation of c-Jun transcription. Because the HTLV-1 Tax protein also up-regulates c-Fos (58), this provides a possible mechanism for a synergistic effect of Tax and ICAM-1 cross-linking on microtubule repolarization.


   FOOTNOTES
 
* This work was supported by the Wellcome Trust (UK) and the Leukemia Research Fund, UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S3. Back

|| To whom correspondence should be addressed: Dept. of Immunology, Wright-Fleming Institute, Imperial College, St. Mary's Campus, Norfolk Place, London W2 1PG, UK. Tel.: 44-20-7594-3730; Fax: 44-20-7402-0653; E-mail: c.bangham{at}imperial.ac.uk.

1 The abbreviations used are: HTLV-1, human T-lymphotropic virus, type 1; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; ICAM-1, intercellular adhesion molecule type 1; MTOC, microtubule organizing center; GFP, green fluorescent protein; Tax protein, HTLV-1 transcriptional transactivator protein; OR, odds ratio; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenyindole; MES, 2-(N-morpholino)ethanesulfonic acid; PKC, protein kinase C; CREB, cAMP-response element-binding protein; TRITC, tetramethylrhodamine isothiocyanate. Back

2 M. Nejmeddine, A. L. Barnard, and C. R. M. Bangham, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Hans-Peter Hauri for providing us with the anti-ERGIC53 mAb and David Derse for the MS9 cell line. We also thank Angelina J. Mosley, Sara E. F. Marshall, Ingrid Muller, and Pascale Kropf for their comments on this paper.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Igakura, T., Stinchcombe, J. C., Goon, P. K., Taylor, G. P., Weber, J. N., Griffiths, G. M., Tanaka, Y., Osame, M., and Bangham, C. R. (2003) Science 299, 1713-1716[Abstract/Free Full Text]
  2. Gundersen, G. G., and Bulinski, J. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5946-5950[Abstract/Free Full Text]
  3. Gotlieb, A. I., Subrahmanyan, L., and Kalnins, V. I. (1983) J. Cell Biol. 96, 1266-1272[Abstract/Free Full Text]
  4. Kupfer, A., Swain, S. L., and Singer, S. J. (1987) J. Exp. Med. 165, 1565-1580[Abstract/Free Full Text]
  5. Kupfer, A., Dennert, G., and Singer, S. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7224-7228[Abstract/Free Full Text]
  6. Geiger, B., Rosen, D., and Berke, G. (1982) J. Cell Biol. 95, 137-143[Abstract/Free Full Text]
  7. Johnson, D. I., and Pringle, J. R. (1990) J. Cell Biol. 111, 143-152[Abstract/Free Full Text]
  8. Stowers, L., Yelon, D., Berg, L. J., and Chant, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5027-5031[Abstract/Free Full Text]
  9. Allen, W. E., Zicha, D., Ridley, A. J., and Jones, G. E. (1998) J. Cell Biol. 141, 1147-1157[Abstract/Free Full Text]
  10. Nobes, C. D., and Hall, A. (1999) J. Cell Biol. 144, 1235-1244[Abstract/Free Full Text]
  11. Kroschewski, R., Hall, A., and Mellman, I. (1999) Nat. Cell Biol. 1, 8-13[CrossRef][Medline] [Order article via Infotrieve]
  12. Hanon, E., Hall, S., Taylor, G. P., Saito, M., Davis, R., Tanaka, Y., Usuku, K., Osame, M., Weber, J. N., and Bangham, C. R. (2000) Blood 95, 1386-1392[Abstract/Free Full Text]
  13. Grassmann, R., Berchtold, S., Radant, I., Alt, M., Fleckenstein, B., Sodroski, J. G., Haseltine, W. A., and Ramstedt, U. (1992) J. Virol. 66, 4570-4575[Abstract/Free Full Text]
  14. Jeang, K. T., Giam, C. Z., Majone, F., and Aboud, M. (2004) J. Biol. Chem. 279, 31991-31994[Free Full Text]
  15. Jacobson, S., Shida, H., McFarlin, D. E., Fauci, A. S., and Koenig, S. (1990) Nature 348, 245-248[CrossRef][Medline] [Order article via Infotrieve]
  16. Kannagi, M., Harada, S., Maruyama, I., Inoko, H., Igarashi, H., Kuwashima, G., Sato, S., Morita, M., Kidokoro, M., and Sugimoto, M. (1991) Int. Immunol. 3, 761-767[Abstract/Free Full Text]
  17. Goon, P. K., Biancardi, A., Fast, N., Igakura, T., Hanon, E., Mosley, A. J., Asquith, B., Gould, K. G., Marshall, S., Taylor, G. P., and Bangham, C. R. (2004) J. Infect. Dis. 189, 2294-2298[CrossRef][Medline] [Order article via Infotrieve]
  18. Bex, F., McDowall, A., Burny, A., and Gaynor, R. (1997) J. Virol. 71, 3484-3497[Abstract]
  19. Semmes, O. J., and Jeang, K. T. (1996) J. Virol. 70, 6347-6357[Abstract]
  20. Tanaka, Y., Masuda, M., Yoshida, A., Shida, H., Nyunoya, H., Shimotohno, K., and Tozawa, H. (1992) AIDS Res. Hum. Retroviruses 8, 227-235[Medline] [Order article via Infotrieve]
  21. Tanaka, Y., Lee, B., Inoi, T., Tozawa, H., Yamamoto, N., and Hinuma, Y. (1986) Int. J. Cancer 37, 35-42[Medline] [Order article via Infotrieve]
  22. Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L., and Hauri, H. P. (1988) J. Cell Biol. 107, 1643-1653[Abstract/Free Full Text]
  23. Etienne-Manneville, S., and Hall, A. (2003) Nature 421, 753-756[CrossRef][Medline] [Order article via Infotrieve]
  24. Shuh, M., Hill, S. A., and Derse, D. (1999) Virology 262, 442-451[CrossRef][Medline] [Order article via Infotrieve]
  25. Weiss, A., Wiskocil, R. L., and Stobo, J. D. (1984) J. Immunol. 133, 123-128[Abstract]
  26. Lomas, M., Hanon, E., Tanaka, Y., Bangham, C. R., and Gould, K. G. (2002) J. Gen. Virol. 83, 641-650[Abstract/Free Full Text]
  27. Smith, M. R., and Greene, W. C. (1990) Genes Dev. 4, 1875-1885[Abstract/Free Full Text]
  28. Robek, M. D., and Ratner, L. (1999) J. Virol. 73, 4856-4865[Abstract/Free Full Text]
  29. Yusuf-Makagiansar, H., Makagiansar, I. T., Hu, Y., and Siahaan, T. J. (2001) Peptides (Elmsford) 22, 1955-1962
  30. Armitage, P., Berry G., and Matthews, J. N. S. (2002) Statistical Methods in Medical Research, 4th Ed, pp. 215-218, Blackwell Scientific Publications, Oxford, UK
  31. Sokal, R. R., and Rohlf, F. J. (2004) Biometry, 3rd Ed., pp. 794-797, W. H. Freeman, New York
  32. Rubbi, C. P., and Rickwood, D. (1996) J. Immunol. Methods 192, 157-164[CrossRef][Medline] [Order article via Infotrieve]
  33. Lowin-Kropf, B., Shapiro, V. S., and Weiss, A. (1998) J. Cell Biol. 140, 861-871[Abstract/Free Full Text]
  34. Sedwick, C. E., Morgan, M. M., Jusino, L., Cannon, J. L., Miller, J., and Burkhardt, J. K. (1999) J. Immunol. 162, 1367-1375[Abstract/Free Full Text]
  35. Etienne-Manneville, S., and Hall, A. (2001) Cell 106, 489-498[CrossRef][Medline] [Order article via Infotrieve]
  36. Alefantis, T., Barmak, K., Harhaj, E. W., Grant, C., and Wigdahl, B. (2003) J. Biol. Chem. 278, 21814-21822[Abstract/Free Full Text]
  37. Smith, M. R., and Greene, W. C. (1992) Virology 187, 316-320[CrossRef][Medline] [Order article via Infotrieve]
  38. Burton, M., Upadhyaya, C. D., Maier, B., Hope, T. J., and Semmes, O. J. (2000) J. Virol. 74, 2351-2364[Abstract/Free Full Text]
  39. Wang, A. H., and Yang, X. J. (2001) Mol. Cell. Biol. 21, 5992-6005[Abstract/Free Full Text]
  40. Berg, P., and Pongratz, I. (2001) J. Biol. Chem. 276, 43231-43238[Abstract/Free Full Text]
  41. Yoshida, M. (2001) Annu. Rev. Immunol. 19, 475-496[CrossRef][Medline] [Order article via Infotrieve]
  42. Jin, D. Y., Spencer, F., and Jeang, K. T. (1998) Cell 93, 81-91[CrossRef][Medline] [Order article via Infotrieve]
  43. Meertens, L., Chevalier, S., Weil, R., Gessain, A., and Mahieux, R. (2004) J. Biol. Chem. 279, 43307-43320[Abstract/Free Full Text]
  44. Chiari, E., Lamsoul, I., Lodewick, J., Chopin, C., Bex, F., and Pique, C. (2004) J. Virol. 78, 11823-11832[Abstract/Free Full Text]
  45. Peloponese, J. M., Jr., Iha, H., Yedavalli, V. R., Miyazato, A., Li, Y., Haller, K., Benkirane, M., and Jeang, K. T. (2004) J. Virol. 78, 11686-11695[Abstract/Free Full Text]
  46. Thyberg, J., and Moskalewski, S. (1999) Exp. Cell Res. 246, 263-279[CrossRef][Medline] [Order article via Infotrieve]
  47. Reddy, T. R., Li, X., Jones, Y., Ellisman, M. H., Ching, G. Y., Liem, R. K., and Wong-Staal, F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 702-707[Abstract/Free Full Text]
  48. Trihn, D., Jeang, K. T., and Semmes, O. J. (1997) J. Biomed. Sci. 4, 47-53[CrossRef][Medline] [Order article via Infotrieve]
  49. Wu, K., Bottazzi, M. E., de la Fuente, C., Deng, L., Gitlin, S. D., Maddukuri, A., Dadgar, S., Li, H., Vertes, A., Pumfery, A., and Kashanchi, F. (2004) J. Biol. Chem. 279, 495-508[Abstract/Free Full Text]
  50. Carlier, M. F., Ducruix, A., and Pantaloni, D. (1999) Chem. Biol. 6, R235-R240[CrossRef][Medline] [Order article via Infotrieve]
  51. Schmitz, A. A., Govek, E. E., Bottner, B., and Van Aelst, L. (2000) Exp. Cell Res. 261, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  52. Maruta, H., He, H., Tikoo, A., Vuong, T., and Nur, E. K. M. (1999) Microsc. Res. Tech. 47, 61-66[CrossRef][Medline] [Order article via Infotrieve]
  53. Palazzo, A. F., Joseph, H. L., Chen, Y. J., Dujardin, D. L., Alberts, A. S., Pfister, K. K., Vallee, R. B., and Gundersen, G. G. (2001) Curr. Biol. 11, 1536-1541[CrossRef][Medline] [Order article via Infotrieve]
  54. Daub, H., Gevaert, K., Vandekerckhove, J., Sobel, A., and Hall, A. (2001) J. Biol. Chem. 276, 1677-1680[Abstract/Free Full Text]
  55. Barnard, A. L., Igakura, T., Tanaka, Y., Taylor, G. P., and Bangham, C. R. (2005) Blood 106, in press
  56. Yamamoto, A., Hara, H., and Kobayashi, T. (1997) J. Neurol. Sci. 151, 121-126[CrossRef][Medline] [Order article via Infotrieve]
  57. Lebedeva, T., Dustin, M. L., and Sykulev, Y. (2005) Curr. Opin. Immunol. 17, 251-258[CrossRef][Medline] [Order article via Infotrieve]
  58. Alexandre, C., and Verrier, B. (1991) Oncogene 6, 543-551[Medline] [Order article via Infotrieve]

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