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Originally published In Press as doi:10.1074/jbc.M511756200 on November 18, 2005

J. Biol. Chem., Vol. 281, Issue 3, 1587-1598, January 20, 2006
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EphB Receptors Regulate Dendritic Spine Morphogenesis through the Recruitment/Phosphorylation of Focal Adhesion Kinase and RhoA Activation*

Michael L. Moeller{ddagger}, Yang Shi{ddagger}, Louis F. Reichardt§1, and Iryna M. Ethell{ddagger}2

From the {ddagger}Division of Biomedical Sciences, University of California Riverside, Riverside, California 92521 and the §Howard Hughes Medical Institute and Department of Physiology, University of California San Francisco, San Francisco, California 94143

Received for publication, October 31, 2005 , and in revised form, November 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Eph receptor tyrosine kinases constitute one of the largest families of transmembrane receptor tyrosine kinases. The family is divided into type A and type B receptor subclasses based on their sequence similarity and ability to bind ephrinA or ephrinB ligands, although some binding interactions between two groups have also been reported (1, 2). Ephephrin interactions are unique because they generate bi-directional signals, with signaling from the Ephs being termed "forward signaling" and signaling from the ephrins being termed "reverse signaling." The intracellular domains of both ephrinBs and Ephs are tyrosine-phosphorylated following tetramerization, and intracellular signaling pathways are activated (3).

EphB-ephrinB interactions are implicated in repulsive axonal guidance, cell migration, topographic mapping, and angiogenesis (4–8). Besides their roles in development, the connection between EphBs and the formation of mature dendritic spines in neurons has been recently demonstrated (9–12). Dendritic spines are small protrusions on the surface of neuronal dendrites that represent the postsynaptic components of most excitatory synapses in the brain. It is believed that a synaptic contact between dendritic filopodia and axon terminals induces morphogenesis of dendritic filopodia into dendritic spines, which is characterized by filopodia shortening and formation of mature mushroom-shaped spines (13). Abnormal development of dendritic spines has been shown to be associated with various neurodevelopmental disorders, among them Rett syndrome, Down's syndrome, Angleman's syndrome, and Fragile X mental retardation (13, 14). The inhibition of EphB forward signaling by the overexpression of a kinase-inactive form of EphB2 (kiEphB2) or triple EphB1/B2/B3 knock-out in cultured hippocampal neurons blocked normal spine formation, resulting in retention of the filopodia (9, 12). In contrast, the activation of EphB receptors resulted in filopodia retraction and shortening (12).

The mechanisms by which EphBs drive spine formation have begun to emerge and may involve the phosphorylation and subsequent clustering of syndecan-2 (9), the recruitment of PDZ proteins (15), as well as the activation of RhoGTPases through direct activation of Rho guanine nucleotide exchange factors (RhoGEFs)3 Intersectin-1, Kalirin-7, and Tiam 1 (10, 11, 16). Also, activation of EphBs results in rapid association with N-methyl-D-aspartate glutamate receptors and N-methyl-D-aspartate receptor tyrosine phosphorylation through activation of the Src family of tyrosine kinases (17–19).

Here we show that activation of EphB2 in cultured hippocampal neurons by its ephrinB ligands results in shortening of dendritic filopodia through the assembly of a protein complex resembling a focal adhesion complex. This complex includes focal adhesion kinase (FAK), Src, Grb2, and paxillin. Furthermore, EphB2 activation leads to the activations of FAK, Src, and paxillin, potentially initiating a number of downstream signaling pathways. Disruption of FAK expression through either Cre-mediated deletion of loxP-flanked fak or siRNA-mediated knock-down of FAK blocks EphB-mediated dendritic filopodia morphogenesis. Moreover, this downstream signaling appears to involve the activation of RhoA because overexpression of constitutively active RhoAV14 mimics the effects of ephrinB2-Fc administration, and EphB-mediated filopodia shortening is blocked by the expression of dominant negative RhoA. Our data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia, and FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The primary antibodies used were: rabbit anti-EphB2 (a gift from E. Pasquale, The Burnham Institute, La Jolla, CA; 0.2 µg/ml for Western blot (WB) and 2 µg/ml for immunocytochemistry (ICC)), goat anti-EphB2 (R & D Systems; 0.2 µg/ml for WB and 2 µg/ml for ICC), mouse anti-FAK (BD Transduction Laboratories; 0.5 µg/ml for WB and 5 µg/ml for ICC), rabbit anti-FAK (Santa Cruz Biotechnologies, Inc.; 1 µg/ml for WB and 4 µg/ml for ICC), rabbit anti-Tyr(P)397 FAK (BIOSOURCE International, Inc.; 0.25 µg/ml for WB and 5 µg/ml for ICC), mouse anti-Grb2 (BD Transduction Laboratories; 0.5 µg/ml for WB and 5 µg/ml for ICC), rabbit anti-Grb2 (Santa Cruz Biotechnologies, Inc.; 1 µg/ml for WB and 4 µg/ml for ICC), rabbit anti-Src (BIOSOURCE International, Inc.; 1 µg/ml for WB and 5 µg/ml for ICC), mouse anti-paxillin (BD Transduction Laboratories; 2 µg/ml for WB and 20 µg/ml for ICC), rabbit anti-Tyr(P)31 paxillin (BIOSOURCE International, Inc.; 1 µg/ml for WB and 5 µg/ml for ICC), rabbit anti-Tyr(P)418 Src (BIOSOURCE International, Inc.; 1 µg/ml for WB and 5 µg/ml for ICC), and HRP-conjugated anti-phosphotyrosine antibody (PY20; BD Transduction Laboratories, Inc.).

Secondary antibodies used for WB were: HRP-conjugated goat anti-rabbit (0.08 µg/ml; Jackson ImmunoResearch Laboratories, Inc.), HRP-conjugated donkey anti-mouse (0.08 µg/ml; Jackson ImmunoResearch Laboratories, Inc.), and HRP-conjugated donkey anti-goat (0.04 µg/ml; Jackson ImmunoResearch, Inc.). Secondary antibodies used for ICC were: Alexa 488-conjugated chicken anti-rabbit IgG, Alexa 488-conjugated donkey anti-mouse IgG, Alexa 594-conjugated chicken anti-mouse IgG (4 µg/ml; Molecular Probes), and fluorescein isothiocyanate-conjugated donkey anti-goat IgG (5 µg/ml; Santa Cruz Biotechnologies, Inc).

Hippocampal Neuron Culture Transfection—Cultures of mouse hippocampal neurons were prepared from mouse E15–16 embryos (wild type and mutant) as previously described with modifications (9). The hippocampal cultures were transiently transfected with GFP at 1–5 days in vitro (DIV) using the calcium phosphate method as previously described (20).

Ligand Induction of EphB2 Receptor—To activate the EphBs in cultured hippocampal neurons, we used ephrinB1-Fc or ephrinB2-Fc (R & D) preclustered with anti-human Fc antibody (Jackson ImmunoResearch Laboratories) prior to the application. To precluster, ephrinB1-Fc, ephrinB2-Fc, or control human Fc fragments were mixed with goat anti-human Fc antibody in a 1:2 ratio and incubated on ice for 1 h before being applied to the cultured neurons. Incubations prior to biochemical assays lasted 15 min, whereas those preceding immunofluorescent labeling lasted 30–60 min. Cell-bound preclustered ephrinB2-Fc/goat anti-human Fc antibody complex was visualized at the cell surface with fluorescein isothiocyanate-conjugated donkey anti-goat IgG.

Two-dimensional Electrophoretic Identification of EphB2-associated Proteins—Briefly, cultured hippocampal neurons were treated at 7 DIV with preclustered ephrinB2-Fc or preclustered human Fc (control) for 15 min. EphB2-associated proteins were immunoprecipitated with anti-EphB2 antibody (9) from Triton-soluble extracts of the treated cells. Immunoprecipitated proteins were separated using a two-dimensional system (Ettan IPGhor; Amersham Biosciences) with 13-cm strips, pH 3–10, for the first dimension and an isoelectric focusing step in conjunction with 6–18% gradient SDS-PAGE mini-gel for the second dimension. The gels were silver-stained in a fashion optimized to be compatible with subsequent mass spectrometry analysis. Spots representing proteins associated with the EphB2 were compared between EphrinB2-Fc-induced versus Fc-treated control cultures. Single protein spots reproducible in three independent experiments were cut, destained, and "in-gel" trypsin-digested, and the peptides were extracted for mass spectrometry on a matrix-assisted laser desorption ionization time-of-flight mass spectrometer DE-STR (PE Biosystems).

Biochemical Assays—For co-immunoprecipitations, cultured hippocampal neurons were treated with 2 µg/ml preclustered ephrinB1-Fc, ephrinB2-Fc, or control Fc for 15 min, and the cell lysates of cultured hippocampal neurons were prepared as previously described (12). The lysates were incubated with 1 µg of one of the following antibodies: anti-EphB2, anti-FAK, anti-Src, anti-Grb2, or anti-paxillin and protein A-Sepharose beads (Sigma) for 4 h at 4 °C. The beads were washed three times with ice-cold lysis buffer. The bound materials were eluted with SDS-PAGE sample buffer, resolved on 8–16% Tris-glycine gels, and immunoblotted with specific primary antibodies followed by incubation with corresponding secondary HRP-conjugated antibody and ECL detection.

To assess the role of Src in the EphB2-mediated phosphorylation of paxillin, 7-DIV cultured hippocampal neurons were pretreated with 0.5 µM PP2 (Calbiochem) or PP3 (Calbiochem) for 45 min prior to induction with preclustered recombinant ephrinB2-Fc or control Fc as described above. The ligand inductions were performed for 15 min, the cultures were lysed and immunoprecipitated against paxillin (1 µg of mouse monoclonal anti-paxillin; BD Transduction Laboratories), and immunoprecipitates were analyzed by immunoblotting with either anti-phosphotyrosine PY20 antibody (1.25 µg/ml; BD Transduction Laboratories), rabbit polyclonal anti-Tyr(P)31 paxillin antibody (1 µg/ml; BIOSOURCE International, Inc.), or total paxillin with mouse monoclonal anti-paxillin antibody (0.75 µg/ml; BD Transduction Laboratories). For quantification purposes, the densitometric values of tyrosine-phosphorylated proteins were normalized to the values of the corresponding total protein bands using Adobe Photoshop.

To assess RhoA activation, preclustered ephrinB2-Fc or control human Fc was applied to 7-DIV hippocampal neuron cultures for 15 min. Following treatment, the cells were lysed, a portion of the lysate was retained for use in the assessment of total RhoA, and GTP-RhoA (active form of RhoA) was immunoprecipitated from the remainder of the lysate using GST-RBD beads (Upstate Cell Signaling Solutions). Following immunoprecipitation, the beads were washed four times, the bound material was eluted with Laemmli buffer, and Western immunoblot analysis was performed. The membranes were probed with anti-Rho antibody (Upstate Cell Signaling Solutions). The level of GTP-RhoA was quantified by densitometry and normalized to total RhoA levels in the lysates (GTP-RhoA/total RhoA).

Immunostaining—Preclustered ephrinB2-Fc or control human Fc was applied to 7-DIV cultures. After 30–60 min of treatment, the cultured cells were fixed in 4% paraformaldehyde in phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and blocked in 5% normal goat serum and 1% bovine serum albumin. The primary antibodies used were: rabbit anti-EphB2, goat anti-EphB2, mouse anti-FAK, mouse anti-paxillin, rabbit anti-Src, rabbit anti-Tyr(P)397 FAK, rabbit anti-Tyr(P)418 Src; and rabbit anti-Tyr(P)31 paxillin. Bound antibodies were detected with secondary antibodies: Alexa 594-conjugated chicken anti-rabbit IgG (4 µg/ml; Molecular Probes), Alexa 488-conjugated donkey anti-mouse IgG, Alexa 488-conjugated chicken anti-mouse IgG (4 µg/ml; Molecular Probes), or fluorescein isothiocyanate-conjugated donkey anti-goat IgG (5 µg/ml; Santa Cruz Biotechnologies, Inc.). Immunostaining was analyzed under a confocal laser scanning microscope (Zeiss LSM 510) or an inverted fluorescent microscope (model TE300; Nikon) as previously described (12, 20). Digital images were imported into Photoshop 7.0 (21).

Image Analysis—The effects of EphB activation on dendritic filopodia were examined in 7-DIV GFP-expressing hippocampal neurons as previously described (20). Briefly, the proximal dendrites (identified as processes extended from the neuronal cell body, at least 1 µm in diameter and also microtubule-associated protein 2-positive) were selected for the analysis of the length and number of dendritic protrusions. Hidden protrusions that protruded toward the back or front of the viewing plane were not counted. 10–15 GFP-expressing neurons were randomly selected for each experimental group, and three or four proximal dendrites/each neuron were analyzed. The length of a protrusion was determined by measuring the distance between its tip and base using Image Pro Plus software. Statistical analysis was performed using Microsoft Excel. Statistical differences between Fc-treated control and ephrinB2-Fc-treated experimental groups of dendritic protrusions were compared by Student's t test. Statistical differences for multiple groups were assessed by one-way ANOVA followed by Newman-Keuls post hoc tests.

For quantification of immunoreactive clusters, inverted fluorescent images (single channel) were analyzed using Image Pro Plus Software. Immunoreactive clusters were defined as 0.1–1.0-µm puncta along the proximal dendrites (with an average pixel intensity at least 50% above that in the adjacent dendritic region) and were counted manually. 10–15 neurons were randomly selected for each experimental group, and three or four proximal dendrites/each neuron were analyzed. The statistical differences between Fc-treated controls and the ephrinB2-Fc-treated group were compared with Student's t test.

Conditional FAK Knock-out—Hippocampal neurons were harvested from E15–16 fak conditional (Cre-loxP) knock-out mice (22) and cultured as previously described (12). The pPGK-Cre vector was obtained from Dr. Marc Schmidt-Supprian (Harvard Medical School). The cultures were transfected at 4–5 DIV using the calcium phosphate method with pEGFP alone or pEGFP + pPGK-Cre. At 7 DIV, the cultures were treated with preclustered Fc or ephrinB2-Fc for 60 min as described under "Ligand Induction of EphB2 Receptor" and processed for indirect immunofluorescence. Dendritic protrusions were visualized by GFP fluorescence, counted, and measured as described under "Image Analysis." FAK knock-out was assessed through FAK immunofluorescent labeling. The double GFP/Cre transfected neurons with no FAK immunoreactive fluorescence above the background were considered FAK knock-outs (see Fig. 5A, arrow). The untransfected counterparts (see Fig. 5A, arrowheads) or control GFP-transfected neurons (see Fig. 5B) showed FAK immunoreactivity.

RhoA Activation or Inhibition—To directly assess the effects of RhoA activation or inhibition on dendritic morphology, 5 DIV hippocampal neurons were double transfected with GFP and Myc-tagged constitutively active RhoA V14 or GFP and hemagglutinin-tagged dominant negative RhoA N19 (both kind gifts of Dr. Katie Defea). Control samples were transfected with GFP only. At 7 DIV, the cultures were treated with preclustered Fc or ephrinB2-Fc for 60 min and processed for indirect immunofluorescence. RhoA V14-transfected cells were labeled with rabbit anti-Myc antibody (Santa Cruz Biotechnologies, Inc.), RhoA N19-transfected cells were labeled with mouse anti-hemagglutinin antibody (clone 12CA5; Roche Molecular Biochemicals). The dendritic protrusions were visualized through GFP fluorescence, counted, and measured as described under "Image Analysis."

siRNA Knockdown of FAK—FAK siRNA was purchased from Santa Cruz Biotechnologies, Inc. (catalog number sc-35353) and transfected into 4-DIV cultured hippocampal neurons using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). Briefly, 1.8 µl of siRNA and 1.2 µl of Lipofectamine 2000/well were reacted together, and transfection was carried out in antibiotic-free Neurobasal medium for 4 h at 37 °C in a 5%CO2, 10%O2 atmosphere. Dendritic spines were visualized by labeling with rhodamine-coupled phalloidin, counted, and measured using Scion Image software. The knock-down efficiency was evaluated through Western immunoblot analysis of FAK expression and FAK immunofluorescent labeling in siRNA-treated versus control samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EphB2 Receptor Recruits FAK, Src, and Grb2 following Ligand-mediated Activation—To identify the signaling molecules that associate with ligand-activated EphB2 in 7-DIV hippocampal neuron cultures following treatment with ephrinB2-Fc, we performed two-dimensional electrophoresis and mass spectrometry analysis using a matrix-assisted laser desorption ionization time-of-flight instrument. A number of proteins were identified in samples from ephrinB2-Fc treated cultures that were not present in Fc-treated cultures. FAK (p125FAK; Swiss Protein Data Base accession number P34152 [GenBank] ) was identified among the proteins associated with ephrinB2-induced EphB2.

To further confirm our mass spectrometry data, we performed reciprocal immunoprecipitation and immunoblotting experiments. For these studies, 7-DIV cultures of hippocampal neurons were treated with either preclustered Fc (control) or preclustered recombinant ephrinB1-Fc or ephrinB2-Fc for 15 min. Cell lysates were subjected to immunoprecipitation against EphB2 and were probed via Western blot for FAK. FAK was found to associate with EphB2 following EphB2 activation, a finding also confirmed by FAK immunoprecipitation followed by EphB2 immunoblotting (Fig. 1A). Further reciprocal co-immunoprecipitation study identified the non-receptor tyrosine kinase Src (Fig. 1B) and the adaptor protein Grb2 (Fig. 1B, right panel) in the EphB2 immunoreactive complex following EphB activation.

Because FAK binds numerous proteins itself, we were also interested in assessing FAK-associated proteins. Reciprocal co-immunoprecipitations identified Src and Grb2 as FAK binding partners following EphB2 activation as well (Fig. 1, B, right panel, and C, respectively). EphB2 activation was confirmed throughout these studies by immunoblotting against phosphotyrosine (Fig. 2A). These data show that EphB2 recruits FAK, Src, and Grb2 following its activation, which suggests that EphB2 might act through a FAK/Src-based mechanism.

FAK and Src Are Activated in Response to EphB2 Activation—With the realization that FAK and Src were components of the EphB2-associated protein complex came the question of how EphB2 activation might be affecting the activation of these kinases. Immunoprecipitations against EphB2, Src, or FAK and immunoblots against phosphotyrosine confirmed increases in EphB2, Src, and FAK phosphorylation following EphB2 activation (Fig. 2, A and B). A robust increase (~48%) in the phosphorylation of FAK on tyrosine 397, a known activator of FAK, was seen in response to treatment with ephrinB2-Fc (Fig. 2B). The increases in FAK phosphorylation on tyrosine 397 were comparable with changes in Src phosphorylation on tyrosine 418 (~40%; Fig. 2, A and B). These data show that levels of specific FAK phosphorylation on tyrosine 397 and Src phosphorylation on tyrosine 418 are significantly increased following ephrinB2-mediated EphB2 activation, indicating that EphB receptors activate FAK and Src following their recruitments.

EphB2, Tyr(P)397FAK, and Tyr(P)418Src Form Discrete Puncta in Dendrites of Hippocampal Neurons Following Treatment with EphrinB2-Fc—Although it was clear from our previous experiments that EphB2, FAK, and Src become associated with one another and activated following treatment with ephrinB2-Fc, we wished to assess whether these associations and activations were occurring specifically in the dendrites. Therefore, we next turned our attention to the cellular distribution of EphB2, FAK, and Src in ephrinB2-induced and control Fc-induced 7-DIV cultured hippocampal neurons through immunocytochemistry. Both EphB2 and active Tyr(P)397FAK formed discrete clusters along the dendritic cell surfaces following treatment with ephrinB2-Fc (Fig. 3, A and C). The distributions of these proteins were relatively diffuse in the absence of EphB2 activation, suggesting that treatment with ephrinB2-Fc drives the formation of these clusters. The double immunolabeling also showed co-localization of EphB puncta and Tyr(P)397FAK clusters as a result of the ephrinB2-mediated activation of EphB2 (Fig. 3E, arrows). Although Src distribution did not appear to change following EphB activation (Fig. 3B), the phosphorylation of Src on tyrosine 418 significantly increased (Fig. 3, D and G), specifically at the sites of EphB clustering (Fig. 3F, arrows). These data show that ephrinB2-mediated activation of EphB receptors induces clustering and co-localization of Tyr(P)397FAK, Tyr(P)418Src, and EphB2 in hippocampal neurons.


Figure 1
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  		FIGURE 1.
EphB2 activation induces its association with FAK, Src, and Grb2 in 7-DIV hippocampal neurons. 7-DIV cultured hippocampal neurons were treated with Fc (2 µg/ml), ephrinB1-Fc (2 µg/ml), or ephrinB2-Fc (2 µg/ml) for 15 min and lysed. The lysates were immunoprecipitated (IP) and immunoblotted (IB) to assess associations between EphB2 and FAK (A), EphB2 and Src (B), FAK and Src (C), EphB2 and Grb2 (D), and FAK and Grb2 (E). The associations between these pairs of proteins were seen to increase following ephrin treatment. The blots are representative of at least three independent experiments.

 
Paxillin Phosphorylation Induced by EphB2 Activation Depends on Src Activity—Recruitment of Src and FAK to the EphB2-associated complex and their increased phosphorylation suggested that other proteins of focal adhesion complexes such as paxillin might also associate with the complex. Immunoprecipitations against FAK and immunoblotting against paxillin showed that FAK association with paxillin was also induced following EphB2 activation (Fig. 2C), thus leading to the conclusion that the protein complex that associates with EphB2 following its activation includes FAK, Grb2, Src, and paxillin. Interestingly, the total tyrosine phosphorylation of paxillin and the specific phosphorylation of paxillin on tyrosine 31 (Figs. 2C and 4, A and B) increased significantly from base levels following EphB2 activation. These increases were ablated by pretreatment with PP2 (a Src inhibitor; Fig. 4, A and B), suggesting that Src serves to activate paxillin following EphB activation. Although paxillin appeared to be relatively abundant in our neurons, and visible clustering was difficult to detect following treatment with ephrinB2-Fc (Fig. 4C), the immunolabeling of hippocampal neurons with an antibody specifically recognizing phosphorylated/activated paxillin (Tyr(P)31pax) showed a significant increase in clustering of phosphorylated paxillin at the sites of EphB clustering following treatment with ephrinB2-Fc (Fig.4D, arrows). These data suggest that EphB2 activation initiates signaling events that include the recruitment and activation of FAK, Src, and paxillin, all of which may lead to the activation of numerous downstream signaling pathways.


Figure 2
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  		FIGURE 2.
EphB activation leads to increases in tyrosine phosphorylation of FAK, Src, and paxillin. A–C, 7-DIV cultured hippocampal neurons were treated with Fc (2 µg/ml), ephrinB1-Fc (2 µg/ml), or ephrinB2-Fc (2 µg/ml) for 15 min, lysed, and immunoprecipitated (IP) against EphB2 (A, left panel), Src (A, right panel), FAK (B and C, left panel), or paxillin (C, right panel). Immunoprecipitates were immunoblotted (IB) against phosphotyrosine (pY), Tyr(P)397FAK, or Tyr(P)418Src. The immunoprecipitates were also immunoblotted against EphB2, Src, FAK, or paxillin as controls for equal loading. D, the level of tyrosine phosphorylation was quantified by densitometry and normalized to total protein level. All of the values are reported as the means of three separate experiments ± S.D. *, p < 0.05; **, p < 0.01 as determined by one-way ANOVA.

 


Figure 3
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  		FIGURE 3.
EphB2 and Tyr(P)397FAK form discrete puncta in dendrites of 7-DIV hippocampal neurons following treatment with ephrinB2-Fc. Treatment with ephrinB2-Fc also increases clustering of active form of Src (pY418Src). 7-DIV hippocampal neurons were treated with Fc (2 µg/ml) or ephrinB2-Fc (2 µg/ml) for 30 min prior to indirect immunofluorescence. A–D, immunodetection of EphB2 (A), Src (B), Tyr(P)397FAK (C), or Tyr(P)Src (D) after treatment with control Fc or ephrinB2-Fc. The images are representative of at least three independent experiments. E, double immunolabeling of Tyr(P)397FAK (red) and EphB receptors (green). F, double immunolabeling of Tyr(P)418Src (red) and EphB receptors (green). The localization of EphB receptors was determined by immunolabeling of cell-bound ephrinB2-Fc (green). Treatment with ephrinB2-Fc induces clustering and co-localization of Tyr(P)397FAK and EphB receptors (arrows in E), as well as additional clustering of active form of Src at the ephrinB2 binding sites along the dendrites (arrows in F). Scale bars, upper panels, 10 µm; lower panels, 5 µm. G, quantitative analysis of the number of EphB2, Tyr(P)397FAK, Tyr(P)418Src, and Tyr(P)31paxillin immunoreactive clusters in dendrites of 7-DIV hippocampal neurons after treatment with Fc or ephrinB2-Fc (B2-Fc). The shaded areas indicate the number of clusters that were detected at the sites of EphB clustering following treatment with ephrinB2-Fc. The means represent the averages of at least three independent experiments, and the error bars indicate S.D. ***, p < 0.001 as determined by t test.

 


Figure 4
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  		FIGURE 4.
EphB activation stimulates the Src-mediated tyrosine phosphorylation of paxillin at tyrosine 31 in 7-DIV cultured hippocampal neurons. A and B, ligand activation of EphB2 leads to increase in the total tyrosine phosphorylation of paxillin (A) and the phosphorylation of paxillin on tyrosine 31 (B). These increases were blocked by pretreatment with the Src inhibitor PP2. The levels of phosphorylation were quantified by densitometry and normalized to total protein levels (phosphotyrosine/total protein ratio). The means represent the averages of at least three independent experiments and are reported as the means ± S.D. *, p < 0.05; **, p < 0.01 as determined by ANOVA. C, double immunolabeling of EphB2 (red) and paxillin (green). The EphB2 (red) but not paxillin (green) forms clusters in 7-DIV cultured hippocampal neurons following EphB2 activation by treatment with ephrinB2-Fc. D, double immunolabeling of Tyr(P)31 paxillin (red) and EphB (green). Treatment with ephrinB2-Fc induces additional clustering of Tyr(P)31 paxillin (red) at the ephrinB2 binding sites (green) along the dendrites (see arrows). Scale bars, 10 µm. IP, immunoprecipitation.

 


Figure 5
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  		FIGURE 5.
Cre-mediated recombination results in loss of FAK expression and causes changes in dendritic morphology of hippocampal neurons. Neurons were obtained from conditional fak mutant mice bearing loxP-flanked fak alleles, co-transfected with pEGFP and pPGK-Cre or pEGFP alone at 4 DIV, and processed for indirect immunofluorescence at 7 DIV. A, the double Cre/GFP-transfected neurons have shown the loss of FAK expression (arrows), whereas untransfected neurons (arrowheads) were immunopositive for FAK (red). GFP fluorescence was used to visualize dendritic morphology. The Cre-transfected neurons exhibited longer, more numerous dendritic filopodia. Scale bars, top panel, 20 µm; middle panel,20 µm; lower panel,5 µm. B, control neurons transfected with GFP only bear normal dendritic morphology (arrowheads) and express detectable levels of FAK (red). The control GFP-transfected neurons possess shorter, less numerous filopodia. C, quantification of dendritic protrusion length. Disruption of FAK expression leads to longer dendritic protrusions. The data represent the mean protrusion length. The error bars indicate S.D. ***, p < 0.001 with one-way ANOVA. D, quantification of dendritic protrusion density. Disruption of FAK expression leads to increased number of dendritic filopodia. The data represent average number of protrusions/10 µm of dendrite. The error bars indicate S.D. **, p < 0.01; ***, p < 0.001 with one-way ANOVA. E, analysis of size distribution curve for dendritic protrusion length in control and FAK knock-out neurons. Disruption of FAK expression leads to a progressive flattening of the size distribution curve for dendritic protrusions. The error bars indicate S.D. KO, knock-out.

 
Disruption of FAK Expression Blocks EphB2-mediated Dendritic Filopodia Shortening—We previously reported that ephrinB2-mediated activation of EphB receptors had pronounced effects on dendritic protrusions in vitro (12). In vivo, this process occurs following the formation of synaptic connections that invariably involve a variety of molecular interactions. To specifically address the role of EphB-ephrinB interaction in spine morphogenesis, we have relied on the use of 7-DIV cell cultures that have not yet formed synaptic connections and have mimicked the effects of endogenous presynaptic ephrinB on EphB activation in the absence of established synapses with recombinant soluble ephrinB2-Fc. The treatment of 7-DIV hippocampal neurons with ephrinB2-Fc resulted in morphological transformation of dendritic filopodia, including the shortening of dendritic filopodia and increased number of shorter, spine-like protrusions. To establish the role of FAK in this process, we assayed whether ephrinB2-mediated EphB activation results in similar changes in dendritic filopodia length in the absence of FAK. FAK elimination in cultured hippocampal neurons derived from conditional fak knock-out mice was achieved by expression of PGK-Cre (kind gift of Dr. Marc Schmidt-Supprian, Harvard Medical School). The hippocampal neurons obtained from fak knock-out mice were double transfected with PGK-Cre and GFP (experimental) or GFP alone (control). FAK knock-out was determined by indirect immunofluorescence against FAK. The double GFP/Cre-transfected neurons with no FAK immunoreactive fluorescence above the background were considered FAK knock-outs (Fig. 5A, arrow), as compared with their untransfected counterparts (Fig. 5A, arrowheads) or control GFP-transfected neurons (Fig. 5B) with positive FAK immunoreactivity. We used GFP fluorescence to visualize dendritic morphology in 7-DIV cultures. The proximal dendrites (identified as processes extended from neuronal cell body, at least 1 µm in diameter and also microtubule-associated protein 2-positive) were selected for the analysis of the length and number of dendritic protrusions. In general, the Cre-transfected neurons exhibited longer, more numerous dendritic protrusions, falling broadly over a range of lengths. By contrast, control GFP-transfected neurons possessed shorter, less numerous filopodia, falling into a relatively more narrow range of length values (Fig. 5, C–E).


Figure 6
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  		FIGURE 6.
Disruption of FAK expression through Cre-mediated recombination leads to an increase in the average length of dendritic protrusions and blocks ephrinB2-Fc-stimulated filopodia shortening in 7-DIV hippocampal neurons. A, confocal images of GFP-labeled FAK knock-out neurons from Cre/GFP-transfected cultures. B, confocal images of GFP-labeled normal neurons from GFP-transfected cultures. Scale bars, top panel,10 µm; bottom panel,5 µm. C, Cre-mediated removal of loxP-flanked fak leads to increases in the average length of dendritic protrusions in Fc-treated neurons and blocks ephrinB2-Fc-mediated filopodia shortening. The data represent the means ± S.D. ***, p < 0.001 with one-way ANOVA. D, Cre-loxP recombinant disruption of FAK expression results in a reduction in the number of short protrusions (<3.0 µm) and an increase in the number of extra long protrusions (>6.0 µm) in controls. Moreover, disruption of FAK expression blocks the effects of ephrinB2 on dendritic filopodia. Although treatment of normal FAK-expressing neurons with ephrinB2-Fc results in a significant increase in the number of short protrusions (<3.0 µm) and a concomitant decrease in the proportion of long filopodia (3.0–6.0 µm), the FAK-deficient neurons show no changes in number of short protrusions or long filopodia after ephrinB2-Fc treatment. E, the distribution curves for the dendritic protrusion length in Fc-treated neurons show higher variability in the protrusion length in the Cre/GFP-expressing neurons (Fc + Cre) than control GFP-transfected neurons (Fc). The control GFP-transfected neurons treated with ephrinB2-Fc (ephrinB2-Fc) show more homogeneous distribution with higher contribution of shorter protrusions, which is disrupted by Cre-recombinant loss of FAK expression (ephrinB2-Fc + Cre).

 
EphB activation by treatment of 7-DIV control neurons with preclustered ephrinB2-Fc causes significant shortening of dendritic filopodia (20–50% decrease), resulting in fewer long filopodia of 3.0–6.0 µm, a large increase in the frequencies of short, spine-like protrusions of <3.0 µm, and a notable compression of the size distribution curve (Fig. 6, B–E). However, disruption of FAK expression through Cre-driven recombination blocked EphB-mediated dendritic filopodia shortening, resulting in a much broader distribution of dendritic protrusion lengths (Fig. 6, A and E). The proportion of long, filopodia-like protrusions (3.0–6.0 µm) remained high in Cre-transfected neurons following treatment with ephrinB2-Fc and was comparable with that of Fc-treated neurons (Fig. 6D). Moreover, Cre-expressing neurons showed many extra long dendritic protrusions of >6.0 µm that were rarely found in normal neurons (~15% in Cre-expressing neurons and only 3% in control GFP-expressing neurons; Fig. 6D).

In addition, FAK knock-downs were produced using commercially available FAK siRNA (Santa Cruz Biotechnologies, Inc.). A partial disruption of FAK expression through RNA interference partially blocked EphB2-mediated filopodia shortening (data not shown). However, the differences between experimental and control groups in the Cre-mediated FAK knock-out experiments were more significant than those of the siRNA-mediated FAK knock-down experiments. These results show that disruption of FAK expression through Cre/loxP-mediated fak deletion or siRNA-mediated FAK knock-down blocks EphB-mediated dendritic filopodia shortening, suggesting that FAK contributes to EphB-mediated dendritic spine formation.

EphrinB2-mediated EphB Activation Induces RhoA Activation—It has been shown that FAK-mediated control of axonal branching occurs, in part, through p190RhoGEF, suggesting that FAK-mediated neurite retraction might proceed through RhoA activation (23). RhoA GTPase has been also shown to underlie Eph-induced filopodia retraction and growth cone collapse during axonal guidance (24). These observations led us to investigate whether this mechanism also underlies the EphB2-mediated morphogenesis of dendritic filopodia. We examined the effect of ligand activation of EphB2 on RhoA activation in 7-DIV hippocampal neurons. The cultured hippocampal neurons were treated with preclustered ephrinB2-Fc or human Fc and assessed for RhoA activation as described under "Experimental Procedures." Western immunoblot analysis of lysates (total RhoA) or GST-RBD immunoprecipitates (active GTP-RhoA) following treatment with either Fc or ephrinB2-Fc revealed a sharp increase in GTP-bound active form of RhoA following EphB activation (Fig. 7A). These results show that EphB activation induces RhoA activation, suggesting that FAK-mediated activation of RhoGEF, which activates RhoA, might also be responsible for the EphB-mediated retraction and shortening of dendritic filopodia.

EphrinB2-mediated EphB Activation Induces Shortening of Dendritic Filopodia through a FAK/RhoA Mechanism—Having shown that EphB activation induces RhoA activation and disruption of FAK expression leads to the ablation of EphB2-mediated shortening of dendritic filopodia, we next examined whether disruption of FAK expression interferes with EphB-mediated RhoA activation. RhoA activity assays following treatment with either Fc or ephrinB2-Fc revealed that EphB activation resulted in substantial increases in the abundance of active RhoA, an effect that was completely ablated by siRNA-mediated FAK knock-down (Fig. 7C). Thus, EphB-mediated activation of RhoA depended on FAK.

Further, we have examined the effects of RhoA activation or inhibition on EphB-mediated shortening of dendritic filopodia. The length of the dendritic filopodia appears to be strongly dependent on the activation status of RhoA, because transfection of cultured hippocampal neurons with a constitutively active RhoA V14 resulted in shortening of dendritic filopodia (Fig. 7, D–G). In fact, the mean dendritic filopodia length in neurons transfected with the constitutively active RhoA V14 was similar to that of control transfected neurons treated with ephrinB2-Fc. Moreover, treatment of RhoA V14-expressing neurons with ephrinB2-Fc did not result in additional shortening of dendritic filopodia.

Although overexpression of a constitutively active RhoA resulted in shortening of dendritic filopodia in cultured hippocampal neurons, overexpression of dominant negative RhoA (RhoA N19) promoted their elongation (Fig. 7, D–G). Moreover, the overexpression of RhoA N19 blocked ephrinB2-Fc-induced shortening of dendritic filopodia. These results reveal that activation of both EphB2 and RhoA cause dendritic filopodia shortening to the same degree, whereas inhibition of RhoA prevents EphB-mediated filopodia shortening and suggests that EphB2-mediated filopodia shortening requires RhoA activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These studies reveal that the activation of EphB2 in cultured hippocampal neurons leads to recruitment of FAK, Src, Grb2, and paxillin, normally considered to be components of focal adhesion complexes. Moreover, EphB2 activation leads to the additional activation of FAK, Src, and paxillin, potentially initiating a number of downstream signaling pathways that are likely to contribute to the assembly of actin filaments in dendritic spines. Furthermore, our data suggest that downstream signaling events initiated by activated FAK might contribute to the EphB-mediated formation of dendritic spines because Cre/loxP-mediated fak knock-out blocks EphB-mediated effects on dendritic protrusion morphology, including filopodia retraction and shortening.

FAK is a non-receptor tyrosine kinase that is widely expressed in different cell types and is typically activated after assembly of integrin-mediated focal adhesions (25). FAK is also expressed in the brain and has been suggested to regulate neurite outgrowth in developing neurons (22). Beggs et al. (22) have reported aberrant dendritic branching in the pyramidal neurons of layers III and V of the cerebral cortex in neuron-specific FAK deletion mutants (22). More recently, FAK has been also shown to function as a negative regulator of axonal development in Purkinje cells and hippocampal neurons controlling extension and pruning of axons (23). Although integrins have been known as the main extracellular regulators of FAK during focal adhesion assembly (26), FAK has recently been suggested to mediate signaling through several cell surface receptors and their ligands including the receptor deleted in colorectal cancer and its soluble ligand netrin (27–29) and Ephs and their ligands, the ephrins (30, 31).

Our studies show that EphB2 activated by ephrinB2-Fc recruits FAK and mediates its activation in 7-DIV cultured hippocampal neurons. Our data also suggest that downstream signaling events initiated by activated FAK contribute to the EphB-mediated shortening of dendritic spine precursors, dendritic filopodia, and their transformations into spine-like protrusions. Previously, inhibition of RhoA or loss of FAK has been shown to reduce neurite retraction, suggesting a relationship between FAK and RhoA signaling (22, 23, 28, 32). Most recently, FAK-mediated control of axonal branching was shown to occur through p190RhoGEF, suggesting that FAK-mediated neurite retraction might proceed through a FAK -> RhoGEF -> RhoA mechanism (23).

This raises the question of whether this mechanism also underlies the EphB2-mediated shortening of dendritic filopodia. Neurons obtained from EphB triple knock-out mice have a high number of long filopodia even in mature 21-DIV cultures (12). In contrast, activation of the EphBs in 7-DIV wild type neurons resulted in filopodia retraction and shortening. Here we show substantial increases in RhoA activity following administration of preclustered ephrinB2-Fc to 7-DIV cultured hippocampal neurons. Furthermore, overexpression of constitutively active RhoA (RhoA V14) mimics the effects of EphrinB2-Fc on filopodia shortening, and EphB-mediated filopodia shortening is blocked by RhoA inhibition. Moreover, both EphB-mediated RhoA activation and filopodia shortening are disrupted by FAK knock-down. These data support the hypothesis that an EphB2 -> FAK -> RhoGEF (p190RhoGEF?) -> RhoA mechanism might also be responsible for the EphB-mediated retraction and shortening of dendritic filopodia.


Figure 7
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  		FIGURE 7.
EphB2-mediated filopodia shortening is dependent on FAK-mediated RhoA activation. A, Western immunoblot analysis of lysates (total RhoA, left panel) or GST-RBD immunoprecipitates (active GTP-RhoA, right panel) following treatment with either Fc or ephrinB2-Fc (B2-Fc) demonstrates a sharp increase in GTP-bound active form of RhoA following EphB activation. B, the levels of total RhoA and GTP-RhoA were quantified by densitometry. The treatment with ephrinB2-Fc (B2-Fc) leads to a 6-fold increase in active GTP-bound RhoA (GTP-RhoA/total RhoA ratio). C, Western immunoblot analysis shows that ephrinB2-induced activation of RhoA is blocked by siRNA-mediated FAK knock-down. D–F, confocal images of GFP-labeled neurons and dendrites of 7-DIV hippocampal neurons transfected with GFP alone (D), GFP plus constitutive active RhoA (RhoA V14, E), or GFP plus dominant negative RhoA (RhoA N19, F) after treatment with control Fc or ephrinB2-Fc. Scale bars, top panel, 10 µm; bottom panel, 5 µm. G, overexpression of constitutively active RhoA (RhoA V14) decreased the average length of dendritic protrusions in Fc-treated neurons. Overexpression of dominant negative RhoA (RhoA N19) blocked ephrinB2-Fc-mediated filopodia shortening. The data represent the means ± S.D. ***, p < 0.001 with one-way ANOVA. H, RhoA activation by overexpression of constitutively active RhoA V14 mimics effects of ephrinB2-Fc resulting in decreased number of long filopodia (3.0–6.0 µm). RhoA inhibition by overexpression of dominant negative RhoA N19 blocks effects of ephrinB2-Fc on filopodia shortening. RhoA N19-positive neurons show significant increases in the number of long filopodia (3.0–6.0 µm) in both Fc-treated and ephrinB2-Fc-treated cultures. I, the distribution curves for the dendritic protrusion length in Fc-treated neurons show higher variability in control GFP-transfected neurons (Fc) than in the neurons expressing constitutive active RhoA (Fc + RhoA V14). The control GFP-transfected neurons treated with ephrinB2-Fc (ephrinB2-Fc) show more homogeneous distribution, with a higher proportion of shorter protrusions, which is disrupted by overexpression of dominant negative RhoA (ephrinB2-Fc + RhoA N19).

 


Figure 8
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  		FIGURE 8.
The EphB2/FAK-associated protein complex and its possible downstream effects on RhoGTPases. Activation of EphB receptors by their ephrinB ligands results in the association of FAK, Grb2, and Src with EphB2. In addition, the frequency of paxillin association with FAK increases. Following assembly of this protein complex, Src phosphorylation increases. Activated Src then accomplishes the phosphorylation of FAK at tyrosine 397 and paxillin at tyrosine 31. Activation of FAK results in RhoA activation and dendritic filopodia shortening. We speculate that Tyr(P)397FAK activates RhoA through p190RhoGEF, resulting in the formation of linear actin filaments that serve as a substrate for the actin-myosin-based shortening and retraction of dendritic filopodia.

 
The published data support a role for RhoA in regulating spine morphology, showing that blocking Rho kinase (ROCK), the downstream target of RhoA, leads to the formation of new, long filopodia with enhanced protrusive motility (33). In contrast, transfection of hippocampal slice cultures with constitutively active RhoA decreases the mean length of dendritic protrusions (34). The Rac1 and Cdc42 RhoGTPases have been also implicated in EphB-mediated formation of dendritic spines through recruitment and activation of the RhoGEFs Kalirin-7, Intersectin-1, and Tiam 1 (10, 11, 16). Both Rac1 and Cdc42 are known to promote the branching of actin filaments, a process that probably contributes to expansion of the spine heads during spine formation and maturation (34–36). EphBs might regulate an initial activation of RhoA through the Tyr(P)397FAK-mediated activation of p190RhoGEF, driving the RhoA-mediated shortening of dendritic filopodia through the formation of linear actin filaments and subsequent actin/myosin-based contraction (Fig. 8). The EphB-mediated activation of Cdc42 and Rac-1, however, might allow for initiation of actin filament branching promoting the formation of the "mushroom" heads, characteristics of mature dendritic spines.

Recruitment of the adaptor protein Grb2, which is capable of associating with both FAK and EphB2, suggests that Grb2 functions as a linker between EphB2 and FAK. Moreover, EphB2 activation also leads to the association of Src with both EphB2 and FAK, thus potentially providing a second molecular link between EphB2 and FAK. Src is known to bind tyrosine 397 of FAK and is responsible for the phosphorylation of both FAK (37) and paxillin (38). Our data support the latter finding, demonstrating that the Src-mediated phosphorylation of paxillin is stimulated by EphB2 activation. The EphB2-mediated activation of paxillin is also significant in that it may tie EphB activation with regulation of different RhoGTPases. The primary role of paxillin is to recruit, organize, and direct the activities of various other proteins, including multiple RhoGEFs and RhoGTPase-activating proteins, which regulate Cdc42, Rac, and other RhoGTPases (39). However, it is unclear from this study which RhoGTPases are being affected by phosphorylated paxillin. The signaling events of downstream of EphB2-mediated paxillin activation in dendritic spines and their role in dendritic spine formation are not clear yet. It will be interesting to determine whether paxillin promotes or inhibits dendritic filopodia extension.

Interestingly, the interactions between the receptor deleted in colorectal cancer and its soluble ligand, netrin-1, have been shown to stimulate growth cone expansion and increase the number of filopodia through a FAK/Src-based mechanism (27, 28, 40). Under other circumstances FAK functions as a negative regulator of axonal branching in part by regulating the function of Rho family GTPases through the activation of p190RhoGEF (23). These disparate effects may be explained by upstream regulators of FAK, which ultimately result in activation of different RhoGEFs and/or RhoGTPase-activating proteins in response to different stimuli. For example, following stimulation with epidermal growth factor, FAK recruits and activates the RhoGEF Vav2, an event that is not seen following integrin activation (41). This phenomenon may be explained by the observation that targeting to focal adhesions via the focal adhesion targeting sequence is the critical determinant of integrin-mediated downstream FAK signaling. Also, the subcellular localization of FAK might be a determinant of FAK function (42). Differences in the upstream receptors (growth factor receptors, integrins, Eph receptors, or receptors deleted in colorectal cancer) to which FAK is recruited might lead to different, possibly divergent effects in downstream signaling. Clearly, future work is needed to characterize how FAK regulates the wide array of signals. FAK clearly plays key roles in various processes related to neuronal development, including those associated with Eph receptors, receptors deleted in colorectal cancer, growth factor receptors, or integrins.


   FOOTNOTES
 
* This work is supported by National Institutes of Health Grants MH67121 (to I. M. E.) and NS19090 (to L. F. R.). 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

1 An Investigator of the Howard Hughes Medical Institute. Back

2 To whom correspondence should be addressed: Division of Biomedical Sciences, University of California Riverside, B601 Statistics Rd., Riverside, CA 92521. Tel.: 951-827-2186; Fax: 951-827-7121; E-mail: iryna.ethell{at}ucr.edu.

3 The abbreviations used are: RhoGEF, Rho guanine nucleotide exchange factor; DIV, days in vitro; FAK, focal adhesion kinase; GFP, green fluorescent protein; Grb2, growth factor receptor-bound protein 2; GTPase, guanosine triphosphatase; ICC, immunocytochemistry; HRP, horseradish peroxidase; siRNA, small interfering RNA; WB, Western blot; GST, glutathione S-transferase; ANOVA, analysis of variance. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Elena Pasquale (The Burnham Institute, San Diego) for the gift of antibodies, Dr. Marc Schmidt-Supprian (Harvard Medical School) for pPGK-Cre plasmid, Dr. Katie DeFea for the gift of RhoA constructs, Olga Itkis for technical assistance, Cecelia Webster for help with two-dimensional gels, and Ronald New for help with matrix-assisted laser desorption ionization time-of-flight analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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