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Previous Article | Next Article 
Molecular and Cellular Biology, January 2003, p. 70-79, Vol. 23, No. 1
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.1.70-79.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Huntsman Cancer Institute and Department of Biology, University of Utah, Salt Lake City, Utah 84112,1 Department of Experimental Pathology, Lund University, 221 85 Lund, Sweden,2 Department of Molecular Genetics and Cell Biology and Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637,3 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021394
Received 8 July 2002/ Returned for modification 15 August 2002/ Accepted 27 September 2002
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ABSTRACT |
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INTRODUCTION |
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-actinin, members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family, Vav, members of the cysteine-rich protein family, the LATS1 tumor suppressor, and members of the p130cas family as binding partners for zyxin (12, 19, 23, 24, 42, 43, 47, 56). The molecular architecture of zyxin is compatible with the protein's postulated role as a scaffold for the assembly of multimeric protein complexes. Zyxin displays three tandemly arrayed LIM domains, double zinc finger structures that support specific protein interactions (37, 47). In addition, zyxin exhibits at least one nuclear export signal that regulates its excursions between the nucleus and the cytoplasm (35, 36). Zyxin also displays four proline-rich ActA repeats that can interact directly with members of the Ena/VASP family (14, 19, 34). Members of the Ena/VASP family are concentrated at focal adhesions, the leading edge of lamellipodia, and filopodial tips (19, 42). Ena/VASP proteins are involved in modulation of actin assembly and organization (4, 6, 30, 41, 48) and are required for normal fibroblast and neuron migration (5, 20). Recent work has illustrated that the proper subcellular targeting of Ena/VASP family members is essential for them to fulfill their normal roles in vivo (1, 5, 6).
The Ena/VASP binding capacity of zyxin has been studied in detail, and several lines of evidence suggest that zyxin plays an important role in localizing members of this family to sites of cell-substratum adhesion. For example, microinjection of peptides that cause displacement of zyxin from focal adhesions also results in loss of Ena/VASP family members from these sites (15). In complementary experiments, targeting of zyxin to membranes or mitochondria leads to recruitment of Ena/VASP proteins (14, 18). Moreover, microinjection of peptide inhibitors that interfere with the binding of Ena/VASP proteins to partners with ActA repeats, such as zyxin, disturbs the subcellular distribution of Ena/VASP proteins and affects cell spreading and cell-cell adhesion (14, 52). Collectively, these studies suggest that failure of Ena/VASP proteins to localize properly results in defects in actin-based cell functions and implicate zyxin as an important contributor to the proper localization of Ena/VASP proteins.
The physiological consequences of disturbing Ena/VASP function have been characterized in vertebrates by analysis of mice in which Mena (mammalian Ena) or VASP functions have been compromised by gene disruption or expression of mutant constructs. Although mice that lack either Mena or VASP are viable and fertile, compromising the functions of murine Ena/VASP family members results in disturbed platelet function, nervous system development, and epithelial sheet formation (3, 21, 29, 52). Here we pursued a reverse genetic strategy to probe the role of zyxin in the mouse. We generated mice that lack zyxin function and analyzed the importance of zyxin for embryonic development and adult functions, with particular attention to processes that are known to depend on members of the Ena/VASP family. Mice that lack zyxin are both viable and fertile and do not display defects in processes that are compromised when either VASP or Mena functions are eliminated. The existence of multiple zyxin family members may obscure our ability to detect the contribution of zyxin to these processes.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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zxyin targeting construct and generation of zyxin-null mice. All mice were maintained in a specific-pathogen-free barrier facility and cared for, handled, and euthanized following guidelines approved by the Institutional Animal Care and Use Committee of the University of Utah. The neomycin resistance selection cassette pKT3lox(A) and the pTK1-pTK2-C targeting vector were provided by Kirk Thomas (University of Utah Transgenic Core Facility). The 19-kb mouse genomic DNA construct was isolated by screening a LambdaFix mouse 129 phage library with a murine zyxin cDNA probe (31).
To generate the targeting construct, a 4.5-kb genomic region that encompasses the translation initiation codon was replaced with a neomycin resistance cassette (neo) for positive selection. Deletion of the 4.5-kb DNA would remove a 5' noncoding exon and the first four coding exons for zyxin. Two thymidine kinase-negative selection cassettes flanked the targeting construct. To assist in cloning and screening, the SpeI site was deleted from the 5' end of the neomycin resistance coding sequence, and a new SpeI site was added to the 3' end of the neomycin resistance cassette in the targeting construct. The linearized zyxin gene targeting construct was electroporated into the 129SVJ GS stem cell line (Genome Systems Inc., St. Louis, Mo.), and recombinant clones were selected.
Positive embryonic stem (ES) cell lines were screened for correct homologous recombination by Southern DNA analysis. Cells from two independently derived ES cell lines (86 and 185) were introduced separately into recipient morulae to generate chimeric mice. The resulting chimeric mice were mated with C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine). Tail DNAs from agouti mice in the resulting litters were analyzed by Southern blot to identify mice in which the targeted allele was transmitted via the germ line. These heterozygous mice served as the founders for two independent zyxin-/- mouse lines (Zyxin-86 and Zyxin-185). The mouse genome database provided information on the intron-exon boundaries and locations within the zyxin gene and the chromosome locations for zyxin gene family members (ncbi.nlm.nih.gov/genome/guide/mouse).
Characterization of ES cell lines and mice. Genomic DNA was prepared from mouse tails by 55°C incubation with 10 mg of proteinase K per ml in 0.5% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8), and 100 mM EDTA (25). For Southern DNA analysis, the DNA was digested with SpeI and then electrophoresed through a 0.8% agarose gel, depurinated (0.25 M HCl for 10 min), followed by capillary transfer (0.4 M NaOH) to nylon filters for 18 h. The DNA was neutralized (20 min in 0.5 M Tris-HCl [pH 7.5], 1.5 M NaCl), washed in 2x SSPE (20 min), dried, and UV cross-linked. The 3' EcoRI-HindIII DNA fragment was labeled with [32P]dCTP (Ready To Go DNA labeling beads; Amersham Pharmacia, Piscataway, N.J.), and used to probe the DNA filters (1 h at 65°C). After washing at 42°C (0.1% SDS with sequential steps of 2x SSPE, 1x SSPE, and 0.1x SSPE), the blot was developed by autoradiography (wild-type zyxin, 11.8 kb; null mutant, 6.2 kb).
For PCR analysis, the genomic DNA was incubated with three primers, WTFzyx (5'-TAC AAG GGC GAA GTC AGG GCG AGT G-3'), WTRzyx (5'-TGG ACG AAG TTT CCG TGT GTT G-3'), and NEOFzyx (5'-GAC CGC TTC CTC GTG CTT TAC-3') and a 30-cycle PCR regimen of 95°C for 60 s, 58°C for 30 s, and 72°C for 50 s, followed by 95°C for 1 min and 72°C for 3 min. The PCR products (wild-type zyxin, 327 bp; null mutant, 473 bp) were electrophoresed on a 1.8% agarose gel and visualized with ethidium bromide.
For analysis of proteins, tissues were dissected from mice (euthanized by Halothane inhalation followed by cervical dislocation), weighed, homogenized in 5 parts H2O with protease and phosphatase inhibitors (100 mM phenylmethylsulfonyl fluoride, 100 mM benzamidine HCl, 1 mg of pepstatin A per ml, 1 mg of phenanthroline per ml, 0.1 M NaF, 0.2 mM sodium orthovanadate), and diluted with an equal amount of Laemmli sample buffer (28). The tissue homogenates were boiled for 5 min, and the DNA was sheared with a 26-gauge needle. Protein extracts (10 µl) were electrophoresed through denaturing 10% polyacrylamide gels and either Coomassie stained or transferred to nitrocellulose by standard methods (51). Western immunoblot analysis was performed by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, N.J.) for detection.
Antibodies. Polyclonal rabbit antizyxin serum B71 was generated by immunizing a rabbit with keyhole limpet hemocyanin coupled to peptide CSPGAPGPLTLKEVEELEQLT (human zyxin amino acids 344 to 363, encompassing the nuclear export sequence). Polyclonal rabbit antizyxin serum B72 was generated by immunizing a rabbit with keyhole limpet hemocyanin coupled to peptide CDFPLPPPPLAGDGDDAEGAL (human zyxin amino acids 70 to 89, encompassing the first ActA repeat). Rabbit immunizations and serum collection (serum samples B71 and B72) were performed at Harlan Bioproducts for Science (Madison, Wis.). Polyclonal rabbit serum B38 (31) is now known to recognize both zyxin and lipoma preferred partner (LPP) and is therefore referred to as a panzyxin antibody. Zyxin antisera were diluted 1:10,000 for Western blot analysis.
Mouse monoclonal antizyxin antibody 1D1 was generated by immunizing mice with bacterially expressed glutathione S-transferase (GST)-human zyxin; the epitope maps to VVAPKPKVNPFRPGDSEPPP (human zyxin amino acids 28 to 48). Mouse monoclonal antibody 12A12 recognizes LIM3 of both zyxin and TRIP6. Mouse monoclonal antibody 164D4 against human zyxin was supplied by Matthias Krause and Jürgen Wehland (45). Polyclonal rabbit serum MP2 against LPP was supplied by Marleen Petit and Wim Van de Ven (38). Polyclonal rabbit serum B65 (supplied by Susanne Kloeker) is directed against mouse TRIP6 peptide KQPEPSRLPQGRSLPR and was affinity purified for these studies. Antibodies directed against laminin (Sigma-Aldrich, St. Louis, Mo.), keratin 1, keratin 5, loricrin (BabCo, Berkeley, Calif.), and fillagrin (BabCo, Berkeley, Calif.) were used as described previously (40).
Platelets. Platelet-rich plasma was prepared from blood collected from wild-type and zyxin-/- mice by established procedures (3). Platelets were exposed to collagen, and the timing of the resulting shape change and aggregation response was measured with a lumiaggregometer (Aggrecorder II PA-3220; Kyoto Kaiichi Kagaku, Kyoto, Japan) as described previously (3, 57). Both collagen and ADP were used as agonists.
Platelet-rich plasma was prepared for Western blot analysis as follows. After anesthetization and cardiac puncture of three wild-type and three zyxin-/- mice, blood was collected into 1.5-ml microcentrifuge tubes containing 20 µl of anticoagulant citrate dextrose (110 mM sodium citrate [pH 7.4], 2.45% dextrose) plus 250 µl of blood per tube. Phosphate-buffered saline containing 4.4 mM EDTA and 0.5% bovine serum albumin was added to the blood-anticoagulant citrate dextrose mixture (400 µl per tube). Centrifugation at 1,000 rpm (Eppendorf microcentrifuge) for 10 min sedimented the red and white blood cells. The supernatant was recentrifuged at 2,700 rpm for 4 min and aspirated. Next, 300 µl of ammonium chloride solution (150 mM ammonium chloride, 1 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.4) was added to each pellet and mixed, followed by the addition of 400 µl of phosphate-buffered saline-EDTA-bovine serum albumin and centrifugation at 2,700 rpm for 4 min. After the platelet pellets were washed twice in phosphate-buffered saline-EDTA-bovine serum albumin and evaluated by microscopic inspection, they were resuspended in SDS sample buffer, electrophoresed (approximately 400,000 platelets per lane), transferred, and probed as described above.
Histology and immunocytochemistry. For general histological analysis to compare wild-type and zyxin-/- mice, tissues were dissected, fixed, embedded, and stained as described previously (9). For examination of brain morphology, silver staining was performed on five adult brains sectioned along the horizontal plane to evaluate possible commissure defects (29). Immunocytochemical analysis of tail skin from newborn mice was performed as described previously (40, 52).
Flow cytometry. Blood samples (0.5 ml) from mice of various zyxin genotypes were incubated with labeled antibodies specific for mouse blood cell markers. The labeled blood cells were then sorted into populations of B cells, T cells (CD4+ and CD8+), granulocytes, and monocytes by flow cytometry (University of Utah Flow Cytometry Core Facility). The blood cell populations were analyzed for standard error means and paired t tests with Prism software (Graph Pad, San Diego, Calif.).
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RESULTS |
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Targeted disruption of zyxin gene. In order to study the role of zyxin in the mouse, we generated a deletion allele by standard methods for engineering gene disruptions by homologous recombination (Fig. 2A) (10, 32). To abolish zyxin function, approximately 4.5 kb of zyxin gene sequence, including coding sequence for the translational initiation codon, was replaced with a neomycin resistance selection cassette. Inspection of the available mouse genome sequence confirmed that a 5' noncoding exon plus four exons encoding amino acids 1 to 333 would be deleted by this strategy. Properly targeted ES cells were identified by Southern blot analysis (Fig. 2B) and were incorporated into recipient morulae. The resulting chimeric animals were bred to C57BL/6 mice to generate heterozygous mice, which were subsequently interbred. The progeny of zyxin+/- intercrosses were genotyped by either Southern blot (Fig. 2C) or PCR (Fig. 2D) analysis.
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As discussed above, one of the best-characterized biochemical activities of zyxin is its ability to bind members of the Ena/VASP family and contribute to the proper subcellular localization of these proteins to sites of cell adhesion. Like zyxin-null animals, mice that lack either VASP or Mena are viable and fertile. However, higher-resolution analysis revealed contributions of Ena/VASP proteins to platelet function (21), axon guidance (29), and epithelial sheet formation (52). Therefore, we examined the consequences of eliminating zyxin function for these three systems in which zyxin is postulated to contribute via its capacity to dock Ena/VASP proteins.
Analysis of zyxin-deficient platelet function. Two groups have generated mice that lack VASP function (3, 21). These mice are viable and fertile but display an altered platelet activation response. Resting platelets are discoid, nonadhesive cells. When exposed to an agonist such as collagen, thrombin, or ADP, they become activated; they rapidly change their shape and begin to aggregate with each other. The precise timing and extent of these two responses to agonist can be monitored by the use of an aggregometer, which detects these two stages as changes in light transmission (57). Although the patterns of expression of Mena and VASP often overlap, platelets provide an example of a cell that expresses primarily VASP (3), enhancing the likelihood of being able to detect a VASP loss-of-function phenotype. Indeed, as can be seen in Fig. 4A, platelets that lacked VASP function underwent shape change precociously but ultimately aggregated to the same extent as their wild-type counterparts (3). In contrast, the activation profiles of wild-type and zyxin-/- platelets were completely coincident, and the platelets achieved comparable aggregation maxima (Fig. 4B), illustrating that zyxin is not required for normal platelet response.
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Analysis of nervous system development. Mice that lack Mena expression are also viable and fertile, but particular deficiencies in brain development were noted in approximately half of the mutant animals examined (29). Specifically, in Mena-/- brains, the corpus callosum and dorsal hippocampal commissure are absent, while the ventral hippocampal commissure is present but reduced in thickness. To evaluate these structures in zyxin-null mice, we compared silver-stained sections of wild-type, zyxin-/-, and Mena-/- brains (Fig. 5). zyxin-null brains did not exhibit defects in any commissure in the brain (ventral or dorsal hippocampal commissures, anterior or posterior commissures, commissures of the superior and inferior colliculi, habenular commissure, or decussation of the superior cerebellar peduncle). Other major fiber tracts such as the internal and external capsules also appeared to be unaffected in zyxin-null brains. Note that the ectopic callosal fibers seen contacting the ventral portion of the ventral hippocampal commissure in Mena-/- brain (arrow) were not seen in the zyxin-/- brain. We did not detect any deficits in axon guidance associated with elimination of zyxin function.
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The first step toward evaluating the possibility that zyxin plays a role in cellular adhesions in the skin was to examine the subcellular distribution of zyxin in the skin. As can be seen in Fig. 6A, zyxin was concentrated at the borders of wild-type epidermal keratinocytes. The basement membrane, which is demarcated by laminin, serves to separate the epidermis from the underlying dermis. The intensity of antizyxin labeling was more prominent at the cell borders of suprabasal layers, consistent with the enhancement of intercellular junctions in these layers. As expected, the zyxin-null mice showed no detectable zyxin protein anywhere in the epidermis (Fig. 6B). No visible abnormalities were noted in the skin or hair of the zyxin-null animals, and the skin of these mice also appeared normal at the histological level (Fig. 6C). The epidermis consisted of the standard morphologically distinct layers, and the hair follicles appeared normal.
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To test whether the biochemical program of differentiation was normal in zyxin-null epidermis, we used specific markers for the distinct layers of the stratified epithelium. The protein keratin 5, which is a marker for the basal layer of the epidermis, was unaltered in the zyxin-null skin (Fig. 6F). Likewise, the spinous layers exhibited the normal differentiation program, as keratin 1, an early marker of differentiation, was expressed immediately adjacent to the basal layer. Terminal differentiation markers of epidermal differentiation such as loricrin (Fig. 6G) and filaggrin (Fig. 6H), which are hallmarks of the granular layer, also showed normal expression patterns.
Altogether, these data suggest that both the adhesive and differentiation programs of the epidermis are unaffected by the lack of zyxin. One possible explanation may be that the absence of phenotypic abnormalities is due to the presence of other zyxin family members which mask the consequences of loss of zyxin function. Indeed, we determined that both an LPP-specific antibody (38) and a panzyxin antibody that recognizes both zyxin and LPP (Fig. 6I) labeled the cell borders of zyxin-null mouse skin, demonstrating the presence of LPP in the epidermal cell junctions.
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DISCUSSION |
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Among zyxin's binding partners, the interaction with Ena/VASP family members is the best characterized. As discussed above, numerous studies have illustrated that zyxin serves as a docking site for Ena/VASP family members and is important for the proper subcellular targeting of these proteins to particular subcellular locations, most notably focal adhesions (1, 14, 15, 19, 34, 42). Therefore, it was anticipated that elimination of zyxin might disturb Ena/VASP function by ablating a key Ena/VASP docking site within cells. Based on that perspective, one might anticipate that elimination of zyxin would have similar consequences as elimination of Mena or VASP.
As was observed of individual targeted VASP and Mena gene disruptions in the mouse, mice that lack zyxin function are viable and fertile. However, mutations in either Mena or VASP or expression of a VASP deletion construct revealed deficiencies in platelet activation, neuronal pathfinding, and epithelial cell-cell adhesion (3, 21, 29, 52). Here we examined the consequences of zyxin gene disruption for each of these processes. Despite the established contribution of zyxin to the docking of Mena/VASP proteins at cell adhesion sites (14), VASP-/- and Mena-/- phenotypes are not recapitulated in the zyxin-/- animals. Ena/VASP family members are complex, multidomain proteins. One possible explanation for the observation that zyxin-/- animals do not display the deficiencies observed in VASP-/- and Mena-/- mice is that phenotypes resulting from loss of Mena and VASP functions are independent of zyxin. For example, it is established that the localization, and therefore presumably the function, of Ena/VASP family members at the distal tips of lamellipodial extensions and at sites of T-cell receptor clustering are independent of zyxin (27, 45).
Alternatively, the consequences of losing zyxin could be masked by the expression of related proteins. Indeed, since initiating our efforts to target the zyxin gene, two proteins that are closely related to zyxin, LPP and TRIP6, were identified (33, 38, 53, 55). We detected no upregulated expression of these proteins in the zyxin-/- mice, so it does not appear that the lack of dramatic phenotype can be attributed to compensatory changes in LPP or TRIP6 expression. However, zyxin family members display similar functional domains and subcellular distributions, and the three zyxin family members exhibit similar tissue distribution patterns (11, 13, 31, 38, 39, 54, 55). Importantly, in the cases of blood platelets, brain, and skin, three tissues which we examined closely for a zyxin-null phenotype, we and others have detected expression of multiple zyxin family members (Fig. 4C and 6I) (13, 31, 39).
This is the first report describing the ablation of a gene in the zyxin family. Although no basal phenotype has yet been observed in the zyxin-/- mice, consequences of eliminating the zyxin gene may emerge if these animals are challenged by physiological stress. In order to assess further the role of the zyxin family in vivo, it will be of interest to disrupt the genes that encode other zyxin family members and to generate compound mutations. There now exist many examples in which deletion of a single member of a gene family has minimal consequences but simultaneous elimination of multiple members has a severe impact on development or adult physiology (17, 22, 50). The availability of a genetic model for studying zyxin function is a critical first step toward elucidating the role of zyxin.
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ACKNOWLEDGMENTS |
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This work was supported by funds provided by the National Institutes of Health to M.C.B. (GM50877) and to F.B.G. (GM58801), the Huntsman Cancer Foundation to M.C.B., a postdoctoral fellowship award by the Cancer Research Institute (New York) to B.B., a predoctoral training grant by the National Institutes of Health to D.N. (T32-CA09602), and the Fonds der Chemischen Industrie to R.F.
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FOOTNOTES |
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Present address: Department of Molecular Medicine, Max Planck Institute for Biochemistry, 82152 Martinsreid, Germany.
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