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Originally published online as doi:10.1189/jlb.0803403 on November 21, 2003

Published online before print November 21, 2003
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(Journal of Leukocyte Biology. 2004;75:233-239.)
© 2004 by Society for Leukocyte Biology

The spreading of B lymphocytes induced by CD44 cross-linking requires actin, tubulin, and vimentin rearrangements

Adriana Sumoza-Toledo and Leopoldo Santos-Argumedo1

Department of Molecular Biomedicine, Centro de Investigación y Estudios Avanzados, I.P.N., México

1 Correspondence: Departamento de Biomedicina Molecular, CINVESTAV-IPN, Av. IPN #2508, Col. Zacatenco, Apartado Postal 14-740, CP 07360, México, D.F., México. E-mail: lesantos{at}mail.cinvestav.mx


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD44 is a polymorphic family of adhesion molecules widely distributed on cells and tissues. CD44 is up-regulated on activated lymphocytes, and it can function as a receptor, mediating rolling and migration. Although it has been demonstrated that anti-CD44 antibodies bound to tissue-culture plates induce multidirectional emission of retractile dendrites ("spreading") in activated murine B lymphocytes, the involvement of cytoskeleton elements in this phenomenon is largely unknown. In this work, it is shown that the generation of dendrites induced by CD44 cross-linking in activated B cells depends on actin, microtubules, and vimentin reorganization. Immunofluorescence analysis showed that dendrite formation began with actin polymerization, and its extension was favored by microtubules and intermediate filaments of vimentin oriented to the polymerized actin. Pretreatment of activated B lymphocytes with cytochalasin E inhibited the dendrites formation; moreover, when cells were treated with this drug at different time points during the dendrite formation process, the stability of the dendrites was affected. In contrast, although the treatment with colchicine and nocodazole (tubulin polymerization inhibitors) inhibited the dendrites formation, it did not inhibit the initial phase of actin polymerization. According to these results, B cell spreading and dendrite formation induced by anti-CD44 antibodies require coordinated rearrangements of actin, microtubules, and vimentin, being the actin cytoskeleton, the most important element that confers stability and drives the morphological changes during B cell spreading, conceivably preparing B lymphocytes for locomotion.

Key Words: cytoskeleton • hyaluronic acid • microtubule-organizing center • motility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytoskeleton has an important role for the dynamic behavior and mechanical stability of the cytoarchitecture, and it is reorganized when cells respond to extracellular stimuli [1 ]. During migration, leukocytes need the rapid conversion of their cytoarchitecture from semirigid to a highly deformable state, as leukocytes must undergo extensive shape changes to penetrate the small potential spaces between endothelial cells. This plasticity in the leukocyte morphology also favors the interactions among cells in an eventual contact during the cellular activation in the spleen or lymph nodes [2 , 3 ].

CD44 is a polymorphic group of class I transmembrane glycoproteins (80–200 kDa) [4 5 6 ], and one of its ligands is hyaluronic acid (HA), a component of extracellular matrix (ECM) [7 ]. CD44 participates in many cellular processes, which include regulation of growth, survival, differentiation and motility of normal cells, and tumoral cell migration and invasion [8 9 10 11 12 13 ]. CD44 null mice have mild abnormalities in myeloid-progenitor migration and bone marrow colonization [14 ] and in the homing of lymphocytes to lymph nodes or the thymus [15 ]. In agreement with a possible role in cellular migration, CD44 is located in the leading edges and lamellipodia of several cell types in vitro. Moreover, CD44 interacts with the actin cytoskeleton through the association with proteins such as ankyrin [16 ] and the ezrin, radixin, moesin family [17 18 19 ], although the significance of these interactions is not entirely understood.

Cross-linking CD44 with monoclonal antibodies (mAb), which recognize different epitopes, including HA domain, induced spreading (characterized by the formation of dendrites and lamellipodia) of preactivated B lymphocytes [20 21 22 23 24 ]. Preactivation with any of the following stimuli, such as lipopolysaccharide (LPS), anti-µ antibodies, anti-CD40 antibodies, or anti-CD38 antibodies plus interleukin-4 (IL-4), prepares the cells for spreading on culture plates precoated with anti-CD44 antibodies [20 21 22 23 24 ]. The spreading of a T cell line by cross-linking CD44 has also been reported [25 ]. These phenomena are dependent on an increase of intracellular calcium [21 ], activation of protein kinase C [21 ], signal transducer and activator of transcription 6 (STAT-6) [23 ], Cdc42, Rac1, Wiskott-Aldrich syndrome protein [24 , 25 ], and actin polymerization [21 , 25 ]. In addition, it has been observed that during T cell spreading, CD44 is colocalized with F-actin in the lamellipodia protusions [25 ].

Conversely, it is known that in response to chemoattractant gradients, motile cells (such as neutrophils and T cells) adopt a polarized morphology, with distinctive leading and trailing edges oriented with respect to the gradient. Actin preferentially accumulates at the leading edge [26 27 28 29 30 ], whereas the microtubules are in the trailing edge (uropod) [31 32 33 ]. Additionally, microtubules and intermediate filaments reorganization has been observed during migration of T lymphocytes [1 ]. However, knowledge of how the components of the cytoskeleton participate in the morphological changes of B lymphocyte upon spreading is very scarce.

The present study was mainly focused on the participation of actin, microtubules, and vimentin in spreading of activated B lymphocytes induced by anti-CD44 antibodies. The results obtained in this work strongly suggest that the generation of dendrites is dependent on the coordinated polymerization of actin, tubulin, and vimentin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female BALB/c mice (6–8 weeks of age) were used in all experiments. They were produced at the Centro de Investigación y Estudios Avanzados (CINVESTAV, México) animal facility, and the Animal Care and Use Committee of CINVESTAV approved all experiments.

Medium
RPMI 1640 (Gibco, Grand Island, NY) was supplemented with 1% nonessential amino acids (Gibco), 5 x 10-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 2 mM glutamine (all from Sigma Chemical Co., St. Louis, MO), and 10% (v/v) fetal calf serum (FCS; Gibco).

B lymphocyte isolation and activation
Mononuclear cells were isolated from spleen by Ficoll-density gradient separation, and B cells were enriched, eliminating T cell by panning using plastic Petri dishes coated with anti-Thy-1 mAb ascites (NIM-R1) [34 ]. For activation (priming), 2 x 106 purified B cells were incubated in 1 ml medium containing LPS from Escherichia coli serotype O55:B5 at 50 µg/ml (Sigma Chemical Co.) plus 10 U/ml murine IL-4 (Genzyme, Cambridge, MA). Cells were incubated for 18–24 h at 37°C in a 24-well plate.

Assay of dendrite formation ("spreading")
Glass slides or polystyrene-96 plates were coated with 20 µg/ml in phosphate-buffered saline (PBS) of NIM-R8 [rat immunoglobulin G (IgG)2a anti-mouse CD44; ref. 20 ] or rat IgG (isotype control) for 1 h at 37°C. The plates were "blocked" with PBS–10% FCS (v/v) for 1 h at 37°C and then washed extensively with medium before use. Activated B cells were transferred without washing to the precoated glass slides or plates and were incubated for 1–2 h at 37°C. Afterwards, they were observed with an inverted microscope at 10x or 40x magnification. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing dendrite-forming cells (DFC) obtained in control wells (100% DFC) with DFC obtained for each condition.

Cells presenting thin, long membrane extensions (usually 2x or 3x the size of their spherical body) were quantified as DFC. In contrast, cells with spherical or pleomorfic morphology but without extension as described above were not considered DFC.

Inhibition of dendrite formation
For these experiments, the cells were divided in three groups: For the first group, the following drugs were added after priming and 30 min before the dendrite formation assay: colchicine and nocodazole (microtubule polymerization inhibitors) and cytochalasin E (actin polymerization inhibitor; all from Sigma Chemical Co.). Dimethyl sulfoxide (DMSO) at 0.05% (Sigma Chemical Co.; representing the highest concentration of the solvent used during any experiment) was used as a toxicity control. Cells with and without treatment were transferred to anti-CD44 antibodies (NIM-R8) and were incubated for 1 h more to induce dendrite formation. Finally, the percentage of DFC was calculated. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing DFC obtained in control wells (DMSO and without treatment; 100%) with DFC obtained for each condition, where no differences were found between DMSO-treated or cells without treatment. In parallel wells, cell viability was also evaluated in each experiment by trypan blue exclusion.

For the second group, cells with dendritic morphology (activated B lymphocytes that were incubated for 1 h with insolubilized anti-CD44 mAb, as described above) were treated with cytochalasin E or DMSO to revert the morphology. After incubation (0–60 min), the cells were fixed and stained for F-actin and ß-tubulin and were then observed under confocal microscopy.

For the third group, cytochalasin E or DMSO was added at the beginning of the spreading (10–60 min), and all of the cultures were further incubated to complete 2 h.

Finally, the cells were fixed and stained for F-actin and ß-tubulin and were then observed under confocal microscopy.

Actin and microtubule localization
Cells in spreading and control cells were washed with PBS, fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.05% Triton X-100 (Sigma Chemical Co.) in PBS at room temperature for 10 min, and then extensively washed with PBS. For staining, 40 µl mouse anti-ß-tubulin antibody (Sigma Chemical Co.), diluted 1:40, or a similar amount of mouse IgG (isotype control) was added to each sample and incubated for 30 min at room temperature. After incubation, cells were washed three times with PBS and incubated for 20 min in the dark with 40 µl fluorescein isothiocyanate (FITC)-anti-mouse IgG (Cappel, West Chester, PA), diluted 1:100. Cells were then counterstained with 40 µl rhodamine-phalloidin (Molecular Probes, Eugene, OR), diluted 1:100, and were incubated in the dark for 15 min at room temperature. Finally, cells were washed with PBS, dried, mounted in Vecta-Shield (Vector Laboratories, Burlingame, CA), and observed by epifluorescence or confocal microscopy.

Intermediate filament localization
Cells in spreading and control cells were washed with PBS, fixed for 5 min at room temperature in 2% paraformaldehyde in PBS, permeabilized with 0.05% Nonidet-P40 (Sigma Chemical Co.) in PBS at room temperature for 5 min, and then extensively washed with PBS. Cells were then incubated with 40 µl goat anti-mouse vimentin (ICN Pharmaceuticals, Costa Mesa, CA) antibody, diluted 1:20, or goat IgG (isotype control) for 2 h at room temperature. After incubation, cells were washed three times with PBS and stained with FITC-anti-goat IgG (Cappel), diluted 1:300, for 30 min in the dark. Finally, cells were washed again with PBS, dried and mounted in Vecta-Shield (Vector Laboratories), and observed by confocal microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of dendrites in activated B lymphocytes by CD44 cross-linking modified the spatial distribution of microtubules, and it was dependent on tubulin polymerization
Dramatic changes in B cell morphology induced by LPS plus IL-4 stimulation and CD44 cross-linking as well as the participation of the actin polymerization in these morphological changes have been reported previously [20 21 22 ]; however, other cytoskeleton elements had not been studied. Therefore, the microtubule participation in the B lymphocyte spreading was analyzed in this work. LPS plus IL-4-prestimulated B cells were transferred to glass slides precoated with anti-CD44 antibodies or rat IgG as isotype control, and then they were incubated for 1 h at 37°C to induce the morphology described as DFC. Control cells or DFC were fixed, indirectly stained with anti-ß-tubulin antibodies, and analyzed by confocal microscopy. Figure 1A shows the distribution of microtubules in control cells: The microtubule bundles were radially distributed in the rounded cells, and the microtubule-organizing center (MTOC) was located in a central position. In contrast, DFC, shown in Figure 1B , had the microtubules reorganized to the dendrites, and MTOC was located near to the growing base of the dendrite. These results suggest that the microtubules contributed to the spreading and dendritic processes of B lymphocytes. To further test the role of microtubules in the spreading of B cells, inhibitory drugs for tubulin polymerization, nocodazole, and colchicine were used (Fig. 1F) . These drugs were independently added to LPS plus IL-4-activated B lymphocytes 30 min before they were transferred to culture plates precoated with anti-CD44 antibodies. The cells were further incubated for 1 h at 37°C and then fixed to evaluate the percentage of DFC. Figure 1F shows that colchicine and nocodazole inhibited the formation of dendrites in a dose-dependent manner. These results suggest that the reconfiguration and integrity of microtubule cytoskeleton are important for the spreading and dendrite formation of activated B lymphocytes.



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  		Figure 1. Coordinated reorganization of actin and microtubules during dendrite formation in CD44-dependent B lymphocyte spreading. Splenic B lymphocytes stimulated overnight with LPS plus IL-4 were transferred to glass slides precoated with monoclonal anti-CD44 antibodies (NIM-R8) or rat IgG (as isotype control), and they were incubated for 1 h at 37°C to induce dendritic processes. Control cells (A) or DFC (B-D) were fixed with paraformaldehyde, permeabilized with Triton X-100, indirectly stained with anti-ß-tubulin antibodies (B) or mouse IgG as isotype control (C), and observed by confocal microscopy (100x original magnification; D is a Nomarski photograph). (E) DFC were fixed at different time points, as stated in each panel, and stained with rhodamine-phalloidin (left panels) and indirectly stained with anti-ß-tubulin antibodies (right panels). Cells were observed by fluorescence microscopy at x100 original magnification. Arrows show the F-actin accumulated in the lamellipodias. (F) Inhibitory drugs (nocodazole and colchicine) were independently added after priming and 30 min before the dendrite assay. Cells were transferred to NIM-R8 and were further incubated for 1 h. Finally, percentage of DFC was evaluated, and cells presenting thin, long membrane extensions (usually 2x or 3x the size of their spherical body) were quantified as DFC. In contrast, cells with spherical or pleomorfic morphology but without extension were considered negative. These experiments were done by triplicate, and the results shown in the graphs represent three independent assays. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing DFC obtained in control wells (DMSO and without treatment; 100%) with DFC obtained for each condition, where no differences between DMSO-treated or cells without treatment were found. Cell viability, evaluated by trypan blue exclusion, was always >95% at the end of each experiment.

 
Coordination between actin and microtubule cytoskeleton was observed during spreading of B lymphocytes induced by CD44 recognition
Organization of actin and microtubules during dendrite formation and spreading was analyzed. For this purpose, DFC were fixed at different time points and stained with rhodamine-phalloidin to detect F-actin and anti-ß-tubulin antibodies for microtubules as described in Materials and Methods; the cells were then analyzed by fluorescence microscopy. It was observed that B lymphocyte spreading began with F-actin accumulation, pushing the membrane to form a lamellipodia. Later, lamellipodia was extended by the reorganization of microtubules allowing the formation of dendrites (Fig. 1E) . This result indicated that actin polymerization preceded microtubule reorganization, where the dendrites were extended by the polymerization of tubulin to the growing end of the cell. At the same time, the MTOC was displaced from its central position to the dendrite base (Fig. 1E) . As a whole, these observations suggest a coordinated polymerization of actin and tubulin to favor the spreading and dendrite formation of activated B lymphocytes.

As a result of these findings, the influence of the organization and integrity of microtubules on actin cytoskeleton, and vice versa during B cell spreading, were analyzed. Therefore, the cells were pretreated with colchicine (tubulin polymerization inhibitor) or cytochalasin E (actin polymerization inhibitor) 30 min before spreading assay, as described previously. Then, the cells were stained with rhodamine-phalloidin and anti-ß-tubulin antibodies, and finally, they were analyzed by confocal microscopy. Figure 2A (top panels) shows that control cells treated with vehicle, F-actin, and microtubules were distributed, as it was observed in previous experiments. When B lymphocytes were pretreated with colchicine, the cells were able to polymerize actin and began the spreading, but the formation of dendrites was not possible in the absence of intact microtubules (middle panels). Finally, when the cells were pretreated with cytochalasin E, they were unable to begin the spreading (Fig. 2A , bottom panels); however, they presented multiple filopodia with a clear staining for ß-tubulin. In addition, the cells accumulated F-actin around the nucleus, and the MTOC was displaced from the center. In control cells and cells treated with colchicine, F-actin was observed as a diffuse but strong staining in lamellipodias, suggesting a fast and active actin polymerization-depolymerization. These results suggest an important role of actin cytoskeleton in the definition of the B lymphocyte morphology, as it forms different cellular structures that may have diverse physiological roles in the cell.



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  		Figure 2. Actin cytoskeleton determinates the stability of the dendrites. (A) B lymphocytes stimulated overnight with IL-4 plus LPS were treated with the vehicle (DMSO; top panels) or the inhibitory drugs colchicine (middle panels) or cytochalasin E (bottom panels), 30 min before the dendrite assay. Cells were then transferred to glass slides precoated with anti-CD44 antibodies and incubated for 1 h at 37°C. DFC were fixed and stained with rhodamine-phalloidin (red) and indirectly stained with anti-ß-tubulin antibodies (green). (B) DFC were exposed (10–60 min) to the inhibitory drug cytochalasin E or the vehicle. After treatment, the cells were fixed and stained with rhodamine-phalloidin (left panels) and indirectly stained with anti-ß-tubulin antibodies (middle panels). Finally, the cells were observed by confocal microscopy at 100x original magnification.

 
To further evaluate how the polymerization of actin may affect the general morphology and in particular, the structure and integrity of the dendrites, DFC were exposed to cytochalasin E, and changes in the morphology were evaluated 10–60 min later. As it can be seen in Figure 2B , the addition of cytochalasin E has a profound and rapid (10 min) effect on the morphology of the cell. The actin was rapidly redistributed in the soma (around the nucleus), losing the microtubules even before the dendrites were completely retracted. Thirty minutes (or 60 min, data not shown) after the addition of the drug, all the cells became rounded, having multiple filopodia containing ß-tubulin, as described above (Fig. 2A , bottom panels). The addition of cytochalasin E in the initial stages of the spreading (10–60 min) led to the same inhibition described above (rounded cells, with multiple filopodia containing ß-tubulin); therefore, data are not shown. These results suggest that the microtubules serve as a support for the elongation of the dendrites; however, this support depends on F-actin cytoskeleton. Thus, actin cytoskeleton was the principal element for B cell spreading, dendrite formation, and stability. As a whole, all these results clearly indicate the requirement of the concerted action of actin and tubulin polymerization to allow the formation of dendrites during the spreading of activated B lymphocytes.

Intermediate filaments were reorganized in dendrite-forming B lymphocytes
Intermediate filaments are expressed in cell type-specific patterns. Besides, these molecules harbor binding sites for high molecular weight proteins interconnecting intermediate filaments with microtubules and microfilaments [1 ]. T lymphocytes express filaments of vimentin that are reconfigured during the polarization of the cell [35 , 36 ]. Thus, it was necessary to verify if intermediate filaments were reorganized during B cell spreading. Therefore, DFC or control cells were fixed, indirectly stained with anti-vimentin antibodies, and analyzed by confocal microscopy. It was observed that intermediate filaments of vimentin were distributed as a coarsely woven cage in round cells (Fig. 3A ). In contrast, filaments were redistributed to the dendrite (Fig. 3B) , with a distribution similar to the microtubules in DFC, as previously shown. These results suggest some interaction or coordination between microtubule bundles and intermediate filaments of vimentin during B cell spreading.



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  		Figure 3. Intermediate filaments are redistributed during CD44-induced spreading of activated B lymphocytes. Splenic B lymphocytes stimulated overnight with IL-4 plus LPS were transferred to glass slides or tissue-culture plates precoated with anti-CD44 mAb (NIM-R8) or rat IgG and were incubated for 1 h at 37°C to induce spreading. Control B cells (A) or dendrite-forming B cells (B and C) were fixed, permeabilized, indirectly stained with rabbit anti-vimentin antibodies (A and B) or rabbit IgG (C, isotype control), and observed by confocal microscopy at 100x original magnification.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The plasticity in the morphology of lymphocytes is crucial for their migration, in an eventual contact with other cells during their activation in the lymph nodes or spleen and at the effector stages of the immune response. The expression of CD44 is increased upon activation of lymphocytes, and its binding to HA is also regulated [10 , 37 ]. There are previous reports that in vitro cross-linking of CD44 on stimulated B lymphocytes [20 21 22 23 24 ] or in a T cell line [25 ] results in modification of their cytoarchitecture. These studies demonstrated the importance of actin polymerization for B and T cell spreading [21 , 25 ]. The morphological changes observed in B lymphocytes were obtained by recognition of different epitopes of CD44, using a variety of mAb such as IM7, NIM-R8, IRAWB14.4, KM114, and KM201 [20 ]. These results suggest that cross-linking CD44 is enough to send signals that promote the reorganization of cytoskeleton.

Dendrite formation and B cell spreading are also observed by cross-linking major histocompatibility complex class II (MHC-II), CD45R, CD23, lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA4), and intercellular adhesion molecule-1 (ICAM-1) [21 , 22 ]. However, cross-linking molecules such as CD22, CD38, CD40, or B cell receptor are unable to induce spreading of activated B lymphocytes [20 ].

In this study, evidences are being provided that dendrite formation and CD44-induced B cell spreading were also dependent on the coordinated rearrangements of actin, microtubules, and intermediate filaments. It was found that B cell spreading began with F-actin accumulation in a zone of the membrane, forming a lamellipodia that later was elongated by reorganization of microtubules and intermediate filaments in this growing zone. Thus, the dendrite was characterized by accumulation of microtubule bundles. It is interesting that the MTOC was observed in the growing base of the dendrites.

Migratory stimuli such as the chemokines stromal cell-derived factor-1{alpha} (SDF-1{alpha}), regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, or monocyte chemoattractant protein-1 (MCP-1) [38 39 40 ] or the recognition of immobilized, recombinant ICAM-1 [32 , 33 ] induce morphological modifications of T and B lymphocytes. During chemotaxis, the amount of cellular F-actin is increased, as well as the accumulation of F-actin at the leading edge of the migratory cell [41 ]. It has also been observed that microtubules are organized in the trailing edge (uropod) [31 32 33 ].

Conversely, when microtubules were disrupted, the percentage of DFC was deeply affected, suggesting that reorganization and active assembly of tubulin are necessary for dendrite formation. B cells treated with tubulin polymerization inhibitors were still able to begin spreading, as actin polymerization was not affected, showing accumulation in lamellipodias as seen in control cells. These results suggest that the enforced locomotion of B lymphocytes may be produced by actin polymerization only. Similar observations have been registered from fibroblasts lacking microtubules [42 ]. However, even when actin polymerization is used to push the membrane, structures such as dendrites are never formed in the absence of tubulin polymerization. In the uropod formation in migrating T cells, microtubules are apparently not necessary [31 ]. It is possible that the B cell spreading morphology corresponds to a migratory phenotype, representing migratory cells during extravasation or in the process of invasion (metastasis) for CD44-expressing tumoral cells.

When B cells were treated with cytochalasin E, they were unable to begin spreading and to form dendrites. Moreover, these cells showed long filopodia based in microtubules and accumulated F-actin around the nuclei. These results suggest that actin cytoskeleton integrity is extremely important to maintain the stability of the dendrites.

Conversely, intermediate filaments are important molecules, as they have binding sites for microtubules and microfilaments interconnecting them [1 ]. They also confer rigidity to circulating lymphocytes and control their deformability [35 , 36 ]. In this work, we showed that vimentin, an intermediate filament, was reconfigured during spreading and dendrite formation of activated B lymphocytes. The distribution of vimentin was similar to the microtubule bundles, and it was accumulated primarily close to the MTOC. These results suggest a coordinated reorganization of vimentin, which may be one of the intermediate filaments connecting microtubule bundles with other elements of the cytoskeleton.

In summary, this paper provides evidence of the concerted participation of actin, microtubules, and intermediate filaments, which act as a dynamic network favoring the morphological modifications of B lymphocytes.

The biological relevance of the modifications in the B lymphocyte cytoskeleton is not clear yet, but the plasticity in the morphology of the cells has been observed in vivo in intact lymph nodes by two-photons microscopy [43 44 45 46 ]. This plasticity may favor the interaction of B lymphocytes with other cells and/or with the ECM, regulating the activation and/or migration of activated B lymphocytes.


   ACKNOWLEDGEMENTS
 
This work was supported by grants (28093N and 33497N) from Consejo Nacional de Ciencia y Tecnología (CONACYT), México. The authors thank Dr. Fernando Navarro for the confocal microscopy facilities and Q. B. P. Blanca Reyes Márquez and Q. F. B. Héctor Romero Ramírez for technical assistance.

Received August 27, 2003; revised October 14, 2003; accepted October 15, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Herrmann, H., Aebi, U. (2000) Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics Curr. Opin. Cell Biol. 12,79-90[CrossRef][Medline]
  2. Anderson, A. O., Anderson, N. D. (1976) Lymphocyte emigration from high endothelial venules in rat lymph nodes Immunology 31,731-748[Medline]
  3. Verschueren, H., De Baetselier, P., Bereiter-Hahn, J. (1991) Dynamic morphology of metastatic mouse T-lymphoma cells invading through monolayers of 10T1/2 cells Cell Motil. Cytoskeleton 20,203-214[CrossRef][Medline]
  4. Ponta, H., Sherman, L., Herrlich, P. A. (2003) CD44: from adhesion molecules to signalling regulators Nat. Rev. Mol. Cell Biol. 4,33-45[CrossRef][Medline]
  5. Screaton, G. R., Bell, M. V., Jackson, D. G., Cornelis, F. B., Gerth, U., Bell, J. I. (1992) Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons Proc. Natl. Acad. Sci. USA 89,12160-12164[Abstract/Free Full Text]
  6. Hirano, H., Screaton, G. R., Bell, M. V., Jackson, D. G., Bell, J. I., Hodes, R. J. (1994) CD44 isoform expression mediated by alternative splicing: tissue-specific regulation in mice Int. Immunol. 6,49-59[Abstract/Free Full Text]
  7. Lesley, J., Hyman, R., Kincade, P. W. (1993) CD44 and its interaction with extracellular matrix Adv. Immunol. 54,271-335[Medline]
  8. Borland, G., Ross, J. A., Guy, K. (1998) Forms and functions of CD44 Immunology 93,139-148[CrossRef][Medline]
  9. Sherman, L., Sleeman, J., Herrlich, P., Ponta, H. (1994) Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression Curr. Opin. Cell Biol. 6,726-733[CrossRef][Medline]
  10. DeGrendele, H. C., Estess, P., Picker, L. J., Siegelman, M. H. (1996) CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway J. Exp. Med. 183,1119-1130[Abstract/Free Full Text]
  11. Bartolazzi, A., Peach, R., Aruffo, A., Stamenkovic, I. (1994) Interaction between CD44 and hyaluronate is directly implicated in the regulation of tumor development J. Exp. Med. 180,53-66[Abstract/Free Full Text]
  12. Sy, M. S., Liu, D., Schiavone, R., Ma, J., Mori, H., Guo, Y. (1996) Interactions between CD44 and hyaluronic acid: their role in tumor growth and metastasis Curr. Top. Microbiol. Immunol. 213,129-153
  13. Lesley, J., Hyman, R., English, N., Catterall, J. B., Turner, G. A. (1997) CD44 in inflammation and metastasis Glycoconj. J. 14,611-622[CrossRef][Medline]
  14. Schmits, R., Filmus, J., Gerwin, N., Senaldi, G., Kiefer, F., Kundig, T., Wakeham, A., Shahinian, A., Catzavelos, C., Rak, J., Furlonger, C., Zakarian, A., Simard, J. J., Ohashi, P. S., Paige, C. J., Gutierrez-Ramos, J. C., Mak, T. W. (1997) CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity Blood 90,2217-2233[Abstract/Free Full Text]
  15. Protin, U., Schweighoffer, T., Jochum, W., Hilberg, F. (1999) CD44-deficient mice develop normally with changes in subpopulations and recirculation of lymphocyte subsets J. Immunol. 163,4917-4923[Abstract/Free Full Text]
  16. Zhu, D., Bourguignon, L. Y. (1998) The ankyrin-binding domain of CD44s is involved in regulating hyaluronic acid-mediated functions and prostate tumor cell transformation Cell Motil. Cytoskeleton 39,209-222[CrossRef][Medline]
  17. Zohar, R., Suzuki, N., Suzuki, K., Arora, P., Glogauer, M., McCulloch, C. A., Sodek, J. (2000) Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration J. Cell. Physiol. 184,118-130[CrossRef][Medline]
  18. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A., Tsukita, S. (1994) ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons J. Cell Biol. 126,391-401[Abstract/Free Full Text]
  19. Legg, J. W., Isacke, C. M. (1998) Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44 Curr. Biol. 8,705-708[CrossRef][Medline]
  20. Santos-Argumedo, L., Kincade, P. W., Partida-Sanchez, S., Parkhouse, R. M. (1997) CD44-stimulated dendrite formation ("spreading") in activated B cells Immunology 90,147-153[CrossRef][Medline]
  21. Partida-Sanchez, S., Garibay-Escobar, A., Frixione, E., Parkhouse, R. M., Santos-Argumedo, L. (2000) CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells Eur. J. Immunol. 30,2722-2728[Medline]
  22. Davey, E. J., Thyberg, J., Conrad, D. H., Severinson, E. (1998) Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes J. Immunol. 160,5366-5373[Abstract/Free Full Text]
  23. Davey, E. J., Greicius, G., Thyberg, J., Severinson, E. (2000) STAT6 is required for the regulation of IL-4-induced cytoskeletal events in B cells Int. Immunol. 12,995-1003[Abstract/Free Full Text]
  24. Westerberg, L., Greicius, G., Snapper, S. B., Aspenstrom, P., Severinson, E. (2001) Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes Blood 98,1086-1094[Abstract/Free Full Text]
  25. Foger, N., Marhaba, R., Zoller, M. (2001) Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells J. Cell Sci. 114,1169-1178[Abstract]
  26. Cassimeris, L., Zigmond, S. H. (1990) Chemoattractant stimulation of polymorphonuclear leucocyte locomotion Semin. Cell Biol. 1,125-134[Medline]
  27. Devreotes, P. N., Zigmond, S. H. (1988) Chemotaxis in eukaryotic cells: a focus on leukocytes and dictyostelium Annu. Rev. Cell Biol. 4,649-686[CrossRef][Medline]
  28. Zigmond, S. H. (1977) Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors J. Cell Biol. 75,606-616[Abstract/Free Full Text]
  29. Downey, G. P. (1994) Mechanisms of leukocyte motility and chemotaxis Curr. Opin. Immunol. 6,113-124[CrossRef][Medline]
  30. Weiner, O. D., Servant, G., Welch, M. D., Mitchison, T. J., Sedat, J. W., Bourne, H. R. (1999) Spatial control of actin polymerization during neutrophil chemotaxis Nat. Cell Biol. 1,75-81[CrossRef][Medline]
  31. Ratner, S., Sherrod, W. S., Lichlyter, D. (1997) Microtubule retraction into the uropod and its role in T cell polarization and motility J. Immunol. 159,1063-1067[Abstract]
  32. Volkov, Y., Long, A., McGrath, S., Ni, E. D., Kelleher, D. (2001) Crucial importance of PKC-ß(I) in LFA-1-mediated locomotion of activated T cells Nat. Immunol. 2,508-514[CrossRef][Medline]
  33. Volkov, Y., Long, A., Kelleher, D. (1998) Inside the crawling T cell: leukocyte function-associated antigen-1 cross-linking is associated with microtubule-directed translocation of protein kinase C isoenzymes ß(I) and {delta} J. Immunol. 161,6487-6495[Abstract/Free Full Text]
  34. Chayen, A., Parkhouse, R. M. (1982) Preparation and properties of a cytotoxic monoclonal rat anti-mouse Thy-1 antibody J. Immunol. Methods 49,17-23[CrossRef][Medline]
  35. Brown, M. J., Hallam, J. A., Colucci-Guyon, E., Shaw, S. (2001) Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments J. Immunol. 166,6640-6646[Abstract/Free Full Text]
  36. Brown, M. J., Hallam, J. A., Liu, Y., Yamada, K. M., Shaw, S. (2001) Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin J. Immunol. 167,641-645[Abstract/Free Full Text]
  37. Lesley, J., Howes, N., Perschl, A., Hyman, R. (1994) Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response J. Exp. Med. 180,383-387[Abstract/Free Full Text]
  38. del Pozo, M. A., Sanchez-Mateos, P., Nieto, M., Sanchez-Madrid, F. (1995) Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway J. Cell Biol. 131,495-508[Abstract/Free Full Text]
  39. Vicente-Manzanares, M., Rey, M., Jones, D. R., Sancho, D., Mellado, M., Rodriguez-Frade, J. M., del Pozo, M. A., Yanez-Mo, M., de Ana, A. M., Martinez-A, C., Merida, I., Sanchez-Madrid, F. (1999) Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 {alpha}-induced lymphocyte polarization and chemotaxis J. Immunol. 163,4001-4012[Abstract/Free Full Text]
  40. Vicente-Manzanares, M., Montoya, M. C., Mellado, M., Frade, J. M., del Pozo, M. A., Nieto, M., de Landazuri, M. O., Martinez, A., Sanchez-Madrid, F. (1998) The chemokine SDF-1{alpha} triggers a chemotactic response and induces cell polarization in human B lymphocytes Eur. J. Immunol. 28,2197-2207[CrossRef][Medline]
  41. Wang, Y. L. (1985) Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling J. Cell Biol. 101,597-602[Abstract/Free Full Text]
  42. Kaverina, I., Krylyshkina, O., Gimona, M., Beningo, K., Wang, Y. L., Small, J. V. (2000) Enforced polarisation and locomotion of fibroblasts lacking microtubules Curr. Biol. 10,739-742[CrossRef][Medline]
  43. Cahalan, M. D., Parker, I., Wei, S. H., Miller, M. J. (2003) Real-time imaging of lymphocytes in vivo Curr. Opin. Immunol. 15,372-377[CrossRef][Medline]
  44. Cahalan, M. D., Parker, I., Wei, S. H., Miller, M. J. (2002) Two-photon tissue imaging: seeing the immune system in a fresh light Nat. Rev. Immunol. 2,872-880[CrossRef][Medline]
  45. Wei, S. H., Miller, M. J., Cahalan, M. D., Parker, I. (2002) Two-photon imaging in intact lymphoid tissue Adv. Exp. Med. Biol. 512,203-208[Medline]
  46. Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. (2002) Two-photon imaging of lymphocyte motility and antigen response in intact lymph node Science 296,1869-1873[Abstract/Free Full Text]



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