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Address correspondence to Stephen Lambert, Dept. of Cell Biology, University of Massachusetts Medical School, 4 Biotech, 377 Plantation St., Suite 326, Worcester, MA 01605. Tel.: (508) 856-8665. Fax: (508) 856-8774. email: Stephen.Lambert{at}umassmed.edu
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Abstract |
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Using a myelinating explant culture system, we have observed that SCs reorganize and polarize microvillar components, such as the ezrin-binding phosphoprotein 50 kD/regulatory cofactor of the sodium-hydrogen exchanger isoform 3 (NHERF-1), actin, and the activated ezrin, radixin, and moesin family proteins before myelination in response to inductive signals. These components are targeted to the SC distal tips where live cell imaging reveals novel, dynamic growth conelike behavior. Furthermore, localized activation of the Rho signaling pathway at SC tips gives rise to these microvillar componentenriched "caps" and influences the efficiency of node formation.
Key Words: glial cells; nodes of Ranvier; myelin sheath; growth cones; microvilli
Abbreviations used in this paper: BME, basal medium Eagle's; DRG, dorsal root ganglion; EBP50, ezrin-binding phosphoprotein 50 kD; ERM, ezrin, radixin, and moesin family proteins; LPA, lysophosphatidic acid; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; ROCK, Rho-associated kinase; SC, Schwann cell; vgsc, voltage-gated sodium channel.
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Introduction |
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The development of the nodes of Ranvier not only involves substantial morphological alterations in the myelinating glial cell but is also hallmarked by the discrete polarization of axonal cell adhesion molecules, cytoskeletal components, and ion channels. Upon myelination, the L1 cell adhesion molecule, the 155-kD isoform of neurofascin, and the cytoskeletal protein ankyrinB are down-regulated and removed from the axonal plasma membrane. Conversely, the 186-kD isoform of neurofascin and Nr-CAM become highly localized to the node in association with ankyrinG and vgsc's (Martini and Schachner, 1986; Kordeli et al., 1995; Davis et al., 1996; Lambert et al., 1997).
The role of axoglial contacts in promoting vgsc clustering at the node remains controversial. vgsc clusters have been observed in the axons of retinal ganglion cells cultured in the presence of oligodendrocyte conditioned media but in the absence of cellular contact (Kaplan et al., 1997). Similarly, observations of vgsc clusters in amyelinated regions of axons from the dystrophic mouse (Deerinck et al., 1997) support the hypothesis that vgsc clustering is independent of glial cell contacts. However, in vitro myelinating culture studies have shown a requirement for Schwann cell (SC) contacts in mediating axonal vgsc clustering (Ching et al., 1999). This is supported by in vivo observations showing that vgsc and other nodal proteins cluster in close proximity to myelin-associated glycoprotein (MAG)-positive SC processes (Vabnick et al., 1996; Lambert et al., 1997) during myelination of the sciatic nerve. Mechanisms underlying these apparently conflicting lines of evidence remain to be determined.
In the mature peripheral nervous system, axoglial contacts at the nodes of Ranvier are established by SC microvilli (Ichimura and Ellisman, 1991), characterized by expression of F-actin (Trapp et al., 1989), ezrin, radixin, and moesin family proteins (ERMs), and ezrin-binding phosphoprotein 50 kD (EBP50; Melendez-Vasquez et al., 2001; Scherer et al., 2001). To investigate a potential role for these microvilli in node formation, we have studied their formation in myelinating dorsal root ganglion (DRG) explant cultures. We have observed that myelinating SCs polarize microvillar components to novel structures at their distal tips (termed caps) in response to signals that induce myelin formation. These caps are enriched in microvillar components including EBP50, actin, and activated phospho-ERMs. Using EBP50-GFP as a probe in live cell imaging experiments, we observed that SC caps are highly dynamic with behavior reminiscent of an axonal growth cone. We also localized the small GTPase RhoA to the SC cap and found that activation of the Rho pathway was linked to cap formation. Together, these results suggest a localized Rho-mediated activation of ERM proteins at the SC cap. Furthermore, the uncoupling of cap formation and myelination interfered with efficient node formation implicating the SC as having a direct role in establishment of the nodes of Ranvier.
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Results |
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EBP50 polarization in less differentiated cultures was then examined. MAG, which is expressed abaxonally by myelinating SCs after 1.5 wraps around its associated axon (Martini and Schachner, 1986, 1988), was used to visualize earlier stages of myelination. After 56 d of induction, EBP50 localized to both heminodal and binary intermediates adjacent to MAG-positive SCs (Fig. 1 M) and in close proximity to axonal clusters of ßIV spectrin (Fig. 1 J), a second marker for the axonal nodal membrane (Berghs et al., 2000; Komada and Soriano, 2002). These results indicate a distinct localization of microvillar components, if not microvilli proper, at mature and developing nodes.
The three ERM proteins (ezrin, radixin, and moesin) are present in SC microvilli (Melendez-Vasquez et al., 2001; Scherer et al., 2001). Hence, we used a pan-ERM antibody in conjunction with our EBP50 antibody to examine microvillar organization in developing SCs. After 6 d in vitro, EBP50 and ERM staining revealed numerous microvilli (Fig. 2, A and B) covering the surface of migrating SC precursors. Mature SCs, characterized by a bipolar phenotype (Jessen et al., 1994), also exhibited numerous microvilli decorating their length (Fig. 2 C).
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Live cell imaging reveals the existence of a novel, dynamic "growth cone"like structure at the tips of myelinating SCs
To visualize the dynamics of cap formation in live cells, an EBP50-GFP fusion construct was used. When transfected into myelinating DRG cultures, this construct was observed to localize to cap structures at SC tips (Fig. 3 A). Moreover, this localization required the ERM binding site of EBP50 (unpublished data). As the binding site for EBP50 is masked on inactive ERM molecules (Gary and Bretscher, 1995; Matsui et al., 1998), these results suggest that localization of EBP50-GFP to SC caps reflects the local activation of ERM molecules at these sites. In support of this idea, EBP50-GFP was found to colocalize with COOH-terminal phospho-ERM staining (Fig. 3 B), indicative of active forms of these proteins (Reczek et al., 1997; Reczek and Bretscher, 1998).
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To determine whether the GFP-remodeling reflected actual changes in the morphology of the SC tip, we studied acutely dispersed SC preparations. This allowed for visualization of individual cells that was not possible without transfection in explants due to the culture complexity. As observed in explant SCs, isolated bipolar cells displayed discrete tips enriched in microvillar components, such as EBP50 and actin (Fig. 4, AH). Furthermore, the tip-localizing ERMs were again found to be enriched in the activated versions of these proteins (Fig. 4, IL), whereas nonphosphorylated ERMs were diffusely present in the SC body and processes (Fig. 4, MP). Concentrations of RhoA were also observed at the SC tips (Fig. 4, QT). Specifically, 79.1 ± 1.7% (data presented as mean ± SEM) of SC tips that were positively immunostained for either EBP50 or phospho-ERM also displayed accumulations of RhoA.
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Downstream effectors implicated in Rho signaling include the Rho-associated kinases (ROCKs), p140mDia, and the phosphatidyl-inositol 4-phosphate 5-kinase (for review see Kaibuchi, 1999). To examine the contribution of the Rho kinases in LPA-induced cap formation, cultures were treated with Y-27632, a pyridine derivative which functions as a specific inhibitor of p160ROCK (ROCKI) and ROCKII (Uehata et al., 1997; Ishizaki et al., 2000). 2 µM of Y-27632 treatment reduced the capacity of LPA to facilitate cap formation to a level similar to that of control cultures maintained in standard feed. Surprisingly, cap formation in cultures maintained in complete myelin feed was reduced by only 35% after 20 µM of Y-27632 treatment (Fig. 6). These results demonstrate that LPA can substitute for serum in cap formation and does so via an Rho-kinasestimulated pathway. However, the inability of 220 µM of Y-27632 to inhibit cap formation in the presence of serum suggests that other pathways may also be operating in this process.
Polarization of microvillar components appears to be an early event in myelination. As microvilli are observed to form contacts with developing nodes of Ranvier, we examined whether SC cap formation was linked to the clustering of proteins at the nodal membrane. Cultures were induced to myelinate under various conditions and myelin segments with associated nodal clusters of ankyrinG detected by triple staining with antibodies to MBP, ankyrinG, and phospho-ERM (Fig. 7, AF). 65.6 ± 3.6% of myelin segments exhibited associated ankyrinG staining at both ends of the segment in cultures treated with both serum and ascorbate. Cultures treated with ascorbate only exhibited 47.2 ± 3.6% of myelin segments with associated ankyrinG staining and a fourfold increase in the number of myelin segments that showed no associated ankyrinG staining (17.4 ± 2.8% of myelin segments vs. 4.1 ± 0.8% of segments in cultures treated with serum and ascorbate). In contrast with a previous paper (Eldridge et al., 1987), the average number of myelin segments formed in each culture appeared independent of serum. However, the addition of serum did result in segments of greater average length than those observed in ascorbate alone (Fig. 7, A and D). No clusters of ankyrinG staining were observed without accompanying phospho-ERM staining (Fig. 7, DF).
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Discussion |
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These observations are summarized in the model shown in Fig. 8. The initial stages of culture establishment include the migration of premyelinating SCs along the radially extended axons emanating from the DRG. These cells appear to be aggressively motile with numerous cell surface microvilli. As the cultures develop, SCs assume their characteristic bipolar myelinating phenotype with microvilli decorating their length. However, treatment of these cultures with serum and ascorbate (particularly serum) results in a reorganization of microvillar components into SC caps. Finally, SCs proceed to form myelin sheaths coupled with the clustering of proteins at the nodes of Ranvier.
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SC caps exhibit morphological and dynamic similarities to axonal growth cones. ERM proteins are also observed in axonal growth cones and their activity appears critical to growth cone function (Paglini et al., 1998). SC caps may also have functional similarities to growth cones, potentially playing a role in axon recognition and segregation during early myelination, as well as node formation at later stages. Of importance would be the identification of SC cap proteins that might participate in these processes. These proteins could well be ERM binding proteins such as CD44, which has been implicated in SC-axon adhesion and neuregulin signaling (Sherman et al., 2000). At the node, the cell adhesion molecules neurofascin and NrCAM have been proposed as candidate proteins for the 4080-nm filaments observed by electron microscopy to link SC microvilli to the nodal membrane (Ichimura and Ellisman, 1991). Perturbing the function of these proteins has been shown to inhibit the clustering of nodal membrane proteins (Lustig et al., 2001). However, potential complementary microvillar receptors for these molecules facilitating SC-axon adhesion remain unknown.
Our data indicate that the addition of serum to DRG explants affects localized changes at SC tips. This might result from concentrations of RhoA that we have observed at the SC tips and/or a potential enrichment of guanine nucleotide exchange factors or guanosine diphosphate dissociation factors at these sites. Given the known effects of Rho on the actin cytoskeleton, it is highly likely that Rho activation at the tip accounts for the dynamic nature of these structures in the presence of serum. As active ERM proteins interact with RhoGDI (guanosine diphosphate dissociation inhibitor; Takahashi et al., 1997), it is likely that ERM activation at the tip will also enhance the formation of active Rho molecules.
As well as local activation of molecules at SC tips, serum may also enhance the movement of vesicle-associated proteins to the tip. For example, exogenous EBP50-GFP is localized to a motile population of punctae in transfected SCs. We have observed that serum causes dramatic changes in SC length and produces significantly longer myelin segments than those formed under serum-free conditions (unpublished data). These findings suggest the potential for functional interdependencies between cap formation, SC elongation and segment length, and effective clustering of nodal membrane proteins.
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Materials and methods |
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Isolated SCs were cultured according to the Brockes method (Brockes et al., 1979). P1 rat pups were killed and their sciatic nerves removed. The nerves were incubated with 0.1% collagenase (Sigma-Aldrich) in L-15 for 30 min followed by 0.1% collagenase with 0.25% trypsin for 30 min. After rinsing with DME + 10% FBS, the nerves were triturated and plated into the same media in an untreated 100-mm culture dish. The next day, 10-5 M 1-(ß-D-arabinofuranosyl) cytosine (AraC; Calbiochem) was added. After 3 d of anti-mitotic treatment, the AraC was washed out and the cells were allowed to recover for 24 h. Anti-Thy1.1 (clone TN-26; 1:100 in DME) and rabbit complement sera HLA-ABC (Sigma-Aldrich; 1:1 in DME) treatment followed to remove residual fibroblasts. Cultures were maintained in Primaria tissue culture flasks (BD Biosciences) with twice weekly feedings of DME + 10% FBS.
EBP50 antibody
An antibody against EBP50 was raised in rabbits for the following study (Covance). A recombinant EBP50-GST fusion was generated (EBP50 cDNA; gift from R. Brian Doctor, University of Colorado Health Sciences Center, Denver, CO) using the pET-41a + vector (Novagen), expressed in bacteria, purified, and used as the immunogen. Antisera were purified using a Talon kit (CLONTECH Laboratories, Inc.), isolated, His-tagged EBP50 (generated using pET-28b + [Novagen]) which was coupled to CNBr Sepharose (Amersham Biosciences). Eluted antibody was dialyzed against PBS followed by 50% glycerol in PBS.
Explant transfection
Explants were transfected using Lipofectamine PLUS (Invitrogen). Cultures were treated for 24 h before transfection with media of choice (Pedraza and Colman, 2000). 1 µg DNA per coverslip was used, and the DNA/PLUS (3.5 µL)/Lipofectamine (2.8 µL) complexes were incubated with the cultures for 34 h in unsupplemented BME. The transfection mixture was then removed. The cultures were rinsed once with BME and returned to the appropriate medium.
Immunostaining
Explants were fixed for 10 min at 4°C in 4% PFA/4% sucrose in PBS. After washing with PBS, cultures were blocked with 10% BSA in PBS-T (PBS + 0.2% Triton X-100) for 1 h at 37°C. Primary antibodies were diluted in 2% BSA in PBS-T and incubated for 12 h at 37°C. Coverslips were washed with PBS-T; then secondary antibodies, also diluted in 2% BSA in PBS-T (1:1,000), were added and incubated for 1 h at 37°C. After washing with PBS-T, followed by PBS alone and a water rinse, coverslips were mounted using a Vectashield mounting medium (Vector Laboratories). SCs were processed similarly with the exceptions of fixation for 20 min at room temperature followed by a 7-min permeabilization using PBS-T and the omission of Triton X-100 from the proceeding steps. Images were captured using IPLab spectrum software (Signal Analytics Corporation) on an upright Axioplan (Carl Zeiss MicroImaging, Inc.) with a Sensys digital camera (Photometrics Limited), and processed using Adobe Photoshop.
Sources of primary antibodies are as follows: anti-neurofilament (Sternberger Monoclonals, Inc.); anti-tubulin (Sigma-Aldrich); anti-ERM and antiphospho-ezrin (Thr567)/radixin(Thr564)/moesin (Thr558) (Cell Signaling Technology, Inc.); anti-MAG (CHEMICON International, Inc.); anti-MBP (Boehringer); anti-RhoA (Santa Cruz Biotechnology, Inc.); Alexa-Fluor 594 phalloidin (Molecular Probes); anti-ßIV spectrin (a gift from M. Komada, Tokyo Institute of Technology, Tokyo, Japan; Komada and Soriano, 2002); anti-ERM (a gift from E. Luna, University of Massachusetts Medical School, Worcester, MA); anti-ankyrinG (Lambert et al., 1997); and anti-EBP50 as described above (EBP50 antibody). All secondary antibodies were purchased from Molecular Probes.
Live cell imaging
Explants or dispersed SCs were grown on either 22-mm square coverslips or 30-mm Rose dishes (World Precision Instruments) and treated as indicated. Preparations were mounted for imaging on an inverted microscope (DMIRE 2; Leica) in a 37°C-heated 5% CO2/95% room air plexiglass environmental chamber. Image acquisition and processing was performed using an extended range cooled CCD camera (ORCA ER; Hamamatsu) and OpenLab software (Improvision).
Online supplemental material
The original time-lapse videos from which the still images presented in Fig. 3 were taken are available for viewing as supplemental material. Video 1 contains time-lapse fluorescence microscopy of an EBP50-GFPtransfected SC maintained in standard feed. Video 2 specifically shows the distal tip of this cell. Video 3 shows time-lapse fluorescence microscopy of an EBP50-GFPtransfected SC after the induction of myelination using serum and ascorbate. Video 4 shows the remodeling EBP50 enriched tip of this cell. Videos are available at http://www.jcb.org/cgi/content/full/jcb.200303039/DC1.
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Acknowledgments |
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These studies were supported by the National Institutes of Health grant RO1NS36637 (to S. Lambert) and by the Neuroscience Training grant 5T32NS07366 (to C. Gatto). We would also like to acknowledge the National Center for Research Resources Shared Equipment grant (1S10RR15775) used to purchase the microscopy system used in the live cell imaging studies presented.
Submitted: 6 March 2003
Accepted: 11 June 2003
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