Report |
Address correspondence to Deanna L. Benson, Box 1065/Dept. of Neurobiology, The Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029. Tel.: (212) 659-5906. Fax: (212) 996-9785. E-mail: deanna.benson{at}mssm.edu; or Stephen R.J. Salton, Dept. of Neurobiology/Box 1065, The Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029. Tel.: (212) 659-5906. Fax: (212) 996-9785. E-mail: stephen.salton{at}mssm.edu
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Abstract |
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Key Words: cell adhesion molecules; neuronal polarity; hippocampus; ezrin; moesin
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Introduction |
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L1 family members exhibit a conserved structural organization consisting of extracellular Ig and fibronectin type III domains, a single transmembrane segment, and a cytoplasmic domain ranging in size from 85 to 148 residues. Binding interactions with the intracellular and extracellular regions have the ability to transduce signals in both directions across the plasma membrane (Hortsch et al., 1998). The extracellular domain of L1 binds a variety of ligands, which include L1 itself, other members of the Ig superfamily, integrins, and extracellular matrix components (Brümmendorf and Rathjen, 1994; Montgomery et al., 1996). Cytoplasmic binding partners include cytoskeletal-associated proteins, protein kinases, and complexes associated with endocytosis and protein trafficking.
In particular, the cytoplasmic domain contains an ankyrin binding site, which couples L1 to the underlying actin cytoskeleton in a manner that can be negatively regulated by phosphorylation (Davis and Bennett, 1994; Hortsch et al., 1998). This interaction is suspected to stabilize L1 within the axonal membrane, an idea supported by the finding that axons eventually degenerate in mutant mice lacking ankyrinB. However, initial axonal outgrowth and differentiation and the targeting of L1 to axons are relatively normal during early development in these mice, indicating ankyrinB is not required for these events (Scotland et al., 1998). Alternative interactions with the cytoskeleton are likely to mediate some L1 functions during the earliest stages of axonal development. The cytoplasmic region also contains a neuronal-specific sequence, RSLE (Arg-Ser-Leu-Glu), that arises from alternative splicing and appears to be essential for sorting L1 to the axonal growth cone (Kamiguchi and Lemmon, 1998). This sequence also participates in the AP2 adaptor-mediated endocytosis of L1 (Kamiguchi et al., 1998), which is required for activation of the mitogen-activated protein kinase signaling pathway that governs L1-mediated neurite outgrowth (Schaefer et al., 1999).
Here, we identify a novel direct interaction between the cytoplasmic domain of L1 and the cytoskeletal-associated protein, ezrin. We further report that disruption of the ezrinactin interaction dramatically alters neuronal morphology, suggesting that the L1ezrinactin interaction may be functionally important in the initial dynamic process of axonal outgrowth and neuronal differentiation.
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Results and discussion |
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ERM family expression peaks during neural differentiation
We examined the developmental time course of ezrin and moesin expression using immunoblots of both cultured hippocampal neurons and NGF-treated PC12 cells (Fig. 2). Ezrin and moesin levels are highest in the youngest (35 d in culture) hippocampal neurons, a time period notable for axonal polarization, outgrowth, and differentiation, and decrease dramatically after the first week in culture. In PC12 cells, ERM expression is highest in cultures treated with NGF for 35 d and decreases after 10 d of NGF treatment. Interestingly, the peak expression of the ERM family in both cell types occurs during neuronal process outgrowth and precedes that reported for ankyrin, which peaks at postnatal day 10 in cerebellar cell cultures (Kunimoto, 1995). These results suggest that the interaction between L1 and ezrin may play a role in the earliest events of neuronal morphogenesis.
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ERM family concentrates in structures undergoing rapid morphogenesis
In hippocampal neurons, immunolabeled ERMs are distributed in axons and dendrites but are most prominent in actin-rich structures like axonal and dendritic growth cones (Goslin et al., 1989; Paglini et al., 1998). Their distribution in cultured fibroblasts and epithelial cells is analogous; ERM proteins are expressed and concentrated at cell surface structures, such as microvilli, ruffling membranes, and cellcell/cellmatrix adhesion sites (Bretscher, 1999). In neurons, occasional foci of ERM labeling are also noted along processes that are sometimes associated with branch points (unpublished data), a pattern consistent with the notion that ERM interactions are critical for the genesis of cell specific morphologies. Unlike L1, the ERM family is not polarized to axons, indicating there is likely to be an alternative binding partner(s) for the ERM family in dendrites.
L1 and the ERM family interact in vivo
To examine interactions between L1 and the ERM family in vivo, antibodies were used to force cluster L1 on the surface of hippocampal neuron axons and on PC12 neurites, at 37°C, in the presence of phenylarsine oxide, a general inhibitor of endocytosis (Doré et al., 1997), or at 12°C (Harder et al., 1998), and the effect on ERM distribution was assessed. All three treatments yield similar results. However, to rule out the contribution of endocytosis quantification data are restricted to 12°C and phenylarsine oxide experiments. In both cell types, antibody-induced capping dramatically changes the labeling pattern for L1 from a smooth continuous distribution to distinct focal clusters (Fig. 3). Double labeling for L1 and the ERM family reveals a precisely coincident pattern of clusters that in the hippocampal neurons is restricted to axons. Digital fluorescence profiles of L1 and ERM-labeled clusters within axons and neurites confirm the distinct colocalization pattern as exhibited by shared peaks in fluorescence intensity (hippocampal neurons correlation coefficient = 0.736, p = <0.0001; PC12 cells correlation coefficient = 0.744, p = <0.0001). PCR analysis was used to confirm that all rat PC12 cells used express only full-length (+RSLE) L1 (Miura et al., 1991; Takeda et al., 1996). Labeling for spectrin, another cortical actin-associated protein, was not altered by the forced clustering of L1 and remained smooth throughout the axon (Fig. 4, AD). L1-forced clustering investigations were also performed in Schwann cells, which express exclusively an L1 variant lacking the cytoplasmic RSLE sequence (Martini et al., 1994; Takeda et al., 1996). In these cells, L1 clustering did not alter ERM protein labeling (Fig. 4, E and F). Together these data provide strong evidence that L1 and ERM proteins interact in vivo, that this interaction is specific for the ERM family of actin binding proteins, and that it requires the RSLE miniexon.
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Materials and methods |
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In vitro binding
"Bait" inserts were cloned into the plasmid pGEX-5 x 2 and all "trapped" cDNAs cloned into the plasmid pET-30b. Protein expression was induced with IPTG, and bacterial proteins were isolated by sonication in PBS (GST proteins) or Tris-imidazole (pET proteins) and centrifugation. GST fusion proteins were bound to glutathione-agarose at room temperature, washed extensively in PBS-0.05% Tween20, and then incubated for 1 h at 4°C with crude extracts of S-tagged proteins. The resin was washed extensively in PBS-Tween at room temperature and eluted with 15 mM reduced glutathione in PBS. Eluates were concentrated and analyzed by SDS-PAGE and immunoblotting using an S protein HRP conjugate (Novagen) and ECL detection.
Cell cultures
Dissociated hippocampal neurons were prepared from hippocampi of embryonic day 18 Sprague Dawley rats as described previously (Benson et al., 1994). Cells were plated at a density of 3,600 cells/cm2 on poly-L-lysine and laminin (25 µg/ml) or L1-Fc substrates (2 µg/ml). L1 substrates were prepared according to De Angelis et al. (1999). The pIg expression plasmid containing an insert of L1-Fc was provided by Dr. J.L. Salzer (New York University School of Medicine). Neurons were maintained in Neurobasal medium containing B27 supplements. PC12 cells were cultured on poly-L-lysine and collagen-coated coverslips in RPMI 1640 containing 10% horse serum and 5% FCS and differentiated using NGF as described previously (Salton et al., 1983). Schwann cells were prepared as described (A.F. Svenningsen and L. Pedraza, personal communication), and their identification was confirmed using immunolabeling for S100 (rabbit polyclonal, 1:250; Dako).
Immunoblots
Cultured hippocampal neurons (325 d growth) were homogenized as described previously (Benson and Tanaka, 1998). PC12 cells were NGF treated and harvested in 2x SDS sample buffer (020 d differentiation). Equal protein amounts were fractionated on 7.5% SDS-polyacrylamide gels then transferred to PVDF paper. Blots were probed with either goat antiezrin (C19, 1:1,000; Santa Cruz Biotechnology, Inc.), rabbit antiezrin (1:1,000; Upstate Biotechnology), rabbit antimoesin (1:1,000; Upstate Biotechnology), or goat anti-L1 (1:1,000; Santa Cruz Biotechnology, Inc.) incubated in HRP-conjugated species-specific secondary antibodies (Amersham Pharmacia Biotech), washed, and visualized with ECL.
Immunohistochemistry
Neuronal cultures were labeled with either rabbit antiezrin (1:1,000; Upstate Biotechnology) or moesin (1:1,000; Upstate Biotechnology) or a mouse IgM monoclonal antibody to the ERM family (clone 13H9) (1:50; a gift from Professor F. Solomon, Massachusetts Institute of Technology, Cambridge, MA [Goslin et al., 1989]). For tissue localization studies, P0 rat pups were transcardially perfused with 4% paraformaldehyde, postfixed overnight in 4% paraformaldehyde, incubated in 7% sucrose/PBS, and then vibratome sectioned (sagittal plane) at 100 µm. Sections were blocked (3% BSA, 2 h) and then double labeled with a rabbit anti-L1 (1:1,000; a gift from Dr. C. Lagenaur, University of Pittsburgh School of Medicine, Pittsburgh, PA [Lemmon et al., 1989]) and the mouse monoclonal to the ERMs (13H9; 1:100). Labeling in both cultures and tissue sections was visualized with antiIgM-FITC (Jackson ImmunoResearch Laboratories) and/or Texas red antirabbit (Vector Laboratories) secondary antibodies. Single labeled sections were used to confirm distribution patterns.
Antibody-induced clustering
Antibodies to L1 were used to force cluster the proteins on cultured hippocampal neurons, NGF-differentiated PC12 cells, and Schwann cells before immunolabeling for ERM proteins. Primary antibodies directed to the extracellular region of L1 (neat supernatant, mouse monoclonal, ASCS4, Developmental Studies Hybridoma Bank) was applied to live cultures for 15 min. Protein clustering was amplified by the application of a biotinylated antimouse secondary antibody (1:200; Vector Laboratories) for 2 h. Cells were then fixed and double labeled for ERM proteins (13H9; 1:20) or spectrin (rabbit polyclonal [240/235], 1:20; Chemicon). (Note that Ezrin specific antibodies used for Western blots could not be used for immunohistochemistry.) L1 labeling was visualized with a streptavidin-Cy3conjugated antibody (Jackson ImmunoResearch Laboratories); ERM labeling was visualized as described above, and spectrin was visualized with an antirabbit FITC (Cappel). To inhibit endocytosis, live incubations were performed either at 12°C or in the presence of 20 µm phenylarsine oxide, both without permeabilization. Confocal scanning laser microscopy was used to investigate all immunolabeling and transfections (Leica TCS-SP [UV]). Correlation coefficients were calculated from fluorescence intensity profiles taken along 2030-µm lengths of 15 labeled axons (hippocampal neurons) or neurites (PC12s) from three different experiments.
Construction of mutant cDNA encoding the NH2-terminal domain of ezrin
The NH2-terminal domain of ezrin was obtained by digesting of the complete coding sequence of human ezrin (a gift from Dr. M. Arpin, Institut Curie, Paris, France) (Algrain et al., 1993) with HindIII and XmaI. This fragment (1,042 bp) was inserted, in frame, into the corresponding sites in the vector pEGFP-N1 (CLONTECH Laboratories, Inc.) and sequenced. Neurons on different substrates (see Results) were transfected at plating using Effectene (QIAGEN) according to the manufacturer's instructions and fixed after 2472 h. Control transfections using pEGFP-F' (CLONTECH Laboratories, Inc.), a farnesylated form of EGFP that is targeted to the plasma membrane, or plasmid without insert were performed concurrently. All processes of six pEGFP-F'- and six N-EzGFPtransfected neurons grown on L1 or laminin or poly-L-lysine substrates were traced and analyzed using the Neurolucida 2000 system (MicroBrightField, Inc.). Endogenous ERM and L1 distribution was labeled in transfected cells using the antibodies described above. Experiments were repeated in three different hippocampal cultures.
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Footnotes |
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* Abbreviations used in this paper: CAM, cell adhesion molecules; ERM, ezrin, radixin, and moesin; GST, glutathione S-transferase.
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Acknowledgments |
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This work was supported by AA12971 (to D.L. Benson and S.R.J. Salton) and a C.J. Martin fellowship (to T.C. Dickson). Confocal microscopy was performed at the MSSM-CLSM core facility supported with funding from a National Institutes of Health shared instrumentation grant (S10 RR0 9145) and a National Science Foundation Major Research Instrumentation grant (DBI-9724504).
Submitted: 20 November 2001
Revised: 25 April 2002
Accepted: 8 May 2002
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