Isolation and characterization of zyxin-null fibroblasts
Murine zyxin is a 564-aa LIM protein () whose binding partner repertoire had suggested a role in actin assembly, cell cycle control, cell motility, and cell signaling. To directly assess the contributions of zyxin to cell function, we have isolated fibroblasts from mice in which the zyxin
gene is disrupted by homologous recombination (Hoffman et al., 2003
). Neither full-length nor truncated zyxin protein is present in the zyxin−/− cells (). Loss of gene function can lead to compensatory up-regulation of other family members. Two proteins that are closely related to zyxin, the lipoma preferred partner (LPP; Petit et al., 2000
) and thyroid receptor-interacting protein 6 (TRIP6; Yi and Beckerle, 1998
), are also expressed in fibroblasts. We detected no alteration in the level of expression of either LPP or TRIP6 in the zyxin−/− cells (), which is consistent with the view that cells do not compensate for the elimination of zyxin by modulating the expression of these highly related proteins.
Figure 1. Immunoblot analysis of zyxin-null fibroblasts. (A) Domains of zyxin include the α-actinin binding site, four ActA Ena/VASP binding repeats, two nuclear export signals (NES), and three LIM domains. Sites of epitopes for zyxin-specific antibodies (more ...)
Some focal adhesion constituents depend on the presence of their binding partners for their stability (Fukuda et al., 2003
). Therefore, we examined whether the levels of zyxin-binding partners were compromised when zyxin was absent and detected no differences in the levels of several zyxin-binding partners (). Likewise, other focal adhesion proteins including vinculin, talin, integrin-linked kinase (ILK), FAK, paxillin, and src were detected at similar levels in wild-type and zyxin−/− cells ().
Elimination of zyxin does not alter mitotic progression
Zyxin displays mitosis-dependent phosphorylation and has been shown to interact directly with the h-warts/LATS1 tumor suppressor, a serine/threonine kinase implicated in cell cycle control (Hirota et al., 2000
). Disturbance of the LATS1–zyxin interaction by use of a dominant-interfering LATS1 truncation product (LATS1 aa 136–700) was shown to interfere with zyxin localization during mitosis and delay mitotic progression, leading to the suggestion that LATS1-dependent recruitment of zyxin to the mitotic apparatus is required for regulated exit from mitosis (Hirota et al., 2000
With the availability of zyxin−/− cells, we were able to test directly whether zyxin is essential for normal mitotic progression. We detected no change in LATS1 protein levels in the zyxin−/− cells (); therefore, zyxin is not essential for LATS1 stability. To look for specific changes in the cell cycle caused by loss of zyxin, flow cytometric analysis of cell cycle progression in exponentially growing wild-type and zyxin−/− cells was used (). No statistically significant differences in cell cycle progression () or the percentage of binucleate cells (not depicted) were observed. These results illustrate that, at least in fibroblasts, mitotic progression and cytokinesis can proceed normally in the absence of zyxin.
Figure 2. The cell cycle is not altered in zyxin-null cells. (A) Flow cytometry profiles of the propidium-iodide DNA content per cell (2 and 4 n) for wild-type (+/+) and zyxin-null (−/−) fibroblasts. (B) Graph of cell cycle distribution (more ...)
Zyxin-null cells display enhanced migration
Many of zyxin's binding partners and biochemical activities suggest that it may play a role in integrin- and actin-linked processes. Therefore, we focused the balance of our analysis on the possible role of zyxin in motility, adhesion, and cytoarchitecture. To determine if loss of zyxin altered cell motility, the behavior of zyxin−/− cells was evaluated using a monolayer wound assay. In blind studies, zyxin−/− fibroblasts were consistently observed to reach the midline of the wound sooner than the wild-type cells (). Quantitative analysis revealed that the zyxin−/− cells displayed a mean migration velocity of 32 μm/h compared with the wild-type migration rate of 18 μm/h (). The enhanced migration of zyxin−/− fibroblasts suggests that zyxin acts as a negative regulator of cell motility.
Figure 3. Monolayer wound assays demonstrate enhanced migration of fibroblasts lacking zyxin. A wound was scraped across monolayer wild-type and zyxin-null fibroblasts, and migration was monitored by time-lapse microscopy. (A) Time points (0, 4, 8, and 12 h) of (more ...)
Zyxin-null cells appear primed for migration, independent of matrix cues
Integrin-dependent adhesion is essential for communicating intracellular contractility to the underlying extracellular matrix and is necessary to drive motility. To explore the contribution of zyxin to cell motility in greater detail, we examined the haptotactic migration of wild-type and zyxin−/− cells toward a variety of integrin ligands using a Boyden chamber transwell migration assay (). We observed a striking difference in haptotactic motility when comparing wild-type and zyxin−/− cells. Except in the cases of 20 μg/ml fibronectin or vitronectin, the zyxin-null cells displayed statistically significant enhancement of migration, relative to wild-type cells.
Figure 4. Boyden chamber transwell assays demonstrate enhanced migration of zyxin-null fibroblasts. (A) Transwell migration (6 h) toward culture medium with 10% FBS on BSA or 20 μg/ml of collagen type I (Col I), collagen type IV (Col IV), fibronectin (Fib), (more ...)
Matrix proteins have differential, concentration-dependent capacities to stimulate cell migration. As can be seen in , migration of wild-type fibroblasts exhibited sensitivity to fibronectin concentration, whereas zyxin-null fibroblasts migrated efficiently independent of fibronectin concentration. The migration of the zyxin-null cells in the absence of matrix cues was statistically indistinguishable from the maximal migration of wild-type cells toward fibronectin. Zyxin-null cells also displayed enhanced migratory potential relative to their wild-type counterparts in a Matrigel invasion assay (). Introduction of a GFP-tagged version of zyxin into the null cells resulted in suppression of cell migration (). GFP-zyxin localized as expected at focal adhesions in the zyxin−/− cells () and was expressed at levels approximating that of zyxin in wild-type cells (). GFP, which did not affect motility, displayed diffuse cellular localization (not depicted). The successful rescue of the null cells by reintroduction of a zyxin transgene illustrates that the migration phenotype observed for the zyxin−/− cells is directly attributable to loss of zyxin.
Zyxin-null cells display enhanced adhesion without any increase in integrin expression
We next tested whether altered cell–substratum adhesion might account for the enhanced motility of the zyxin−/− cells. Zyxin-null fibroblasts were more adherent than wild-type fibroblasts when plated on a variety of extracellular matrix proteins (), and reexpression of GFP-zyxin in null cells resulted in reduced adhesion (). One possible explanation for the enhanced cell adhesion observed in the zyxin−/− cells could be elevated integrin expression. However, immunoblot analysis revealed no difference in integrin levels or subunit expression ().
Figure 5. Adhesion to ECM proteins is enhanced in zyxin-null fibroblasts, but integrin expression is unchanged. (A) Wild-type and zyxin-null fibroblasts were evaluated for adhesion to vitronectin, laminin, collagen IV, and fibronectin (5 μg/ml; 25 min). (more ...)
Distinct profile of tyrosine-phosphorylated proteins in zyxin-null fibroblasts
The behavior of zyxin-null fibroblasts on integrin ligands suggested that cells lacking zyxin might somehow be primed for integrin-dependent adhesion and migration. One molecular hallmark of the integrin activation state is enhanced tyrosine phosphorylation of proteins such as FAK, which is a tyrosine kinase that colocalizes with zyxin at focal adhesions. Therefore, we compared the subcellular distributions and molecular mass profiles of tyrosine-phosphorylated proteins in wild-type and zyxin-null cells. Both wild-type and zyxin-null cells displayed phosphotyrosine (pY) associated with focal adhesions (), and Western immunoblot analysis of pY-containing proteins revealed no apparent difference in the region where FAK migrates (). However, the wild-type cells displayed a 75-kD tyrosine-phosphorylated protein (pY75) that showed reduced abundance in zyxin-null cells. In contrast, we observed an 80-kD tyrosine-phosphorylated protein (pY80) in the zyxin-null cells that was not evident in the wild-type cells. Using two-dimensional (2D) gel electrophoresis of wild-type and zyxin-null cell lysates, followed by Western immunoblot with antiphosphotyrosine antibody, both pY75 and pY80 were resolved as a cluster of isoelectric point variants ranging from ~6.5 to 8.0 ().
Figure 6. An altered tyrosine-phosphorylated protein in zyxin-null cells is identified as the actomyosin regulator caldesmon. Indirect immunofluorescence of pY-containing proteins in wild-type (A) and zyxin-null (B) fibroblasts. (C) Immunoblot analysis of equivalent (more ...)
The actomyosin regulator caldesmon is altered in zyxin-null cells
Mass spectrometric (LC-MS/MS) sequence analysis of the protein found four pY75 2D gel spots derived from wild-type cells and four pY80 2D gel spots derived from zyxin-null cells, such as those in , and identified all eight tyrosine-phosphorylated spots as caldesmon (). Caldesmon is an actomyosin regulator that influences stress fiber formation, cell contractility, and cell motility (Wang, 2001
). There are two major isoforms of caldesmon derived by alternative splicing (): nonmuscle or low molecular weight caldesmon (l-caldesmon) and smooth muscle or high molecular weight caldesmon (h-caldesmon; Wang, 2001
). Our sequence results are consistent with recovery of l-caldesmon. With the exception of sequence encoded by exon 1′, which was only obtained for pY75 isolated from wild-type cells, sequence from all other classical l-caldesmon exons (Hayashi et al., 1992
; Guo and Wang, 2005
) were represented and were nearly identical for pY75 and pY80 ().
In wild-type mouse fibroblasts, a caldesmon-specific antibody detected a prominent 75-kD immunoreactive band, as well as a minor 80-kD immunoreactive band, which is consistent with l-caldesmon, whereas zyxin-null fibroblasts displayed only the slower migrating 80-kD species (). A similar quantitative shift to a slower mobility species occurred for the 140-kD h-caldesmon in the zyxin-null fibroblasts, as seen in a longer exposure (, right). This shift in caldesmon mobility was detected in multiple zyxin-null primary fibroblast isolates, as well as in immortalized zyxin-null fibroblast lines (unpublished data). Moreover, the caldesmon mobility shift was observed in cells derived from zyxin knock-out mice generated from two independently targeted embryonic stem cell lines (not depicted) and in a variety of tissues, including lung, bladder, and spleen, which were isolated directly from the zyxin-null mice (). The slower migrating caldesmon isoform is evident only in tissues derived from homozygous mutant animals, suggesting that complete loss of zyxin is required to promote the change in caldesmon.
Posttranslational modification does not account for the caldesmon isoform variation observed in the zyxin-null cells
Phosphorylation can affect a protein's electrophoretic mobility and, in the case of caldesmon, is known to regulate its ability to control contractility and integrin-dependent motility (Wang, 2001
). We tested directly whether l-caldesmon from wild-type and zyxin-null cells was differentially phosphorylated on Ser497 and Ser527, two ERK-dependent phosphorylation sites implicated in regulation of caldesmon function (D'Angelo et al., 1999
). Both the 75- and 80-kD caldesmon species were recognized with the phosphospecific antibody (). For a more global assessment of whether differential phosphorylation might be responsible for the caldesmon mobility difference we have observed, we treated cell lysates from wild-type and zyxin-null cells with phosphatase and assayed caldesmon and control paxillin protein mobility by SDS-PAGE (). Although the phosphatase treatment resulted in the collapse of paxillin bands indicative of dephosphorylation, no impact on caldesmon mobility was observed, suggesting that phosphorylation is unlikely to account for the altered caldesmon mobility seen in the zyxin-null cells. To directly test whether caldesmon is subject to differential posttranslational modification that affects its mobility, we transfected wild-type and zyxin-null cells with a his-caldesmon cDNA expression construct and monitored mobility of the expressed protein with Western immunoblot. The mobility of the his-tagged caldesmon was indistinguishable in wild-type and zyxin-null cells, whereas the endogenous caldesmon exhibited the mobility shift (). Collectively, our results suggest that differential posttranslational modification of caldesmon is not likely to be responsible for the altered caldesmon mobility we observe in the zyxin-null cells. Re-expression of zyxin does not restore caldesmon to its wild-type mobility (), which illustrates that the phenotypes observed in the zyxin-null cells are linked to loss of zyxin and are not attributable to alteration of caldesmon.
Figure 7. Caldesmon protein in wild-type and zyxin-null fibroblasts. Fibroblasts were grown for 24 h in high (10% FBS) and low (0.5% FBS) serum, and then collected for immunoblot analysis. (A) Phospho-ser527/789-specific antibody detected caldesmon from both wild-type (more ...)
Depletion of Mena and VASP from focal adhesions of zyxin-null cells
Zyxin is concentrated in the focal adhesions of wild-type fibroblasts (); no immunoreactivity is detected in zyxin-null cells (). Elimination of zyxin did not lead to changes in focal adhesion morphology or number, as assessed by qualitative inspection of vinculin localization (). The distributions of several other focal adhesion markers, including FAK and paxillin, also appeared unperturbed in the zyxin-null cells (unpublished data). In contrast, we detected a dramatic diminution in the localization of the zyxin-binding partners, Mena and VASP, at focal adhesions (; and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200512115/DC1
) even though their cellular levels were unchanged (). In double-labeling experiments, it was evident that Mena colocalized with vinculin at focal adhesions of wild-type cells () and was depleted from vinculin-rich focal adhesions in zyxin-null cells (). Thus, the altered Mena localization was not attributable to loss of focal adhesion structures. Expression of GFP-zyxin in the null cells restored localization of Mena to vinculin-rich focal adhesions (). Similar results were obtained for VASP (unpublished data). These observations are consistent with a major role for zyxin in localizing Ena/VASP family members at focal adhesions. Residual Mena and VASP accumulation at focal adhesions in the zyxin−/− cells likely reflects the presence of other focal adhesion proteins that can dock Ena/VASP family members. For example, both vinculin and LPP are present in focal adhesions and have the capacity to bind Ena/VASP proteins (Brindle et al., 1996
; Petit et al., 2000
). Interestingly, LPP was detected more prominently at focal adhesions of zyxin-null cells, as compared with cocultured wild-type cells ().
Figure 8. Localization of proteins in wild-type and zyxin-null fibroblasts demonstrate the importance of zyxin for proper localization of Mena and VASP. Protein localization in wild-type (+/+) and zyxin-null (−/−) cells was evaluated (more ...)
Zyxin-null cells fail to build robust actin stress fibers when stimulated with jasplakinolide
The mislocalization of Ena/VASP proteins and the alteration of caldesmon observed in the zyxin-null cells raised the possibility that actin filament assembly or organization might be disturbed when zyxin function is compromised. Likewise, previous work involving expression of a dominant-negative zyxin deletion fragment (Nix et al., 2001
), RNA interference (Harborth et al., 2001
), and exposure of cells to mechanical stimuli (Yoshigi et al., 2005
) suggested that zyxin might be required for actin cytoskeletal assembly or maintenance. To assess this possibility, we compared the actin cytoskeletons of wild-type and zyxin-null cells using phalloidin staining (). The presence of actin filament arrays in the zyxin-null fibroblasts illustrated that zyxin was not absolutely essential for establishment and maintenance of actin stress fibers. To explore whether we might detect an effect of eliminating zyxin when cells were challenged to reorganize actin arrays, we compared the response of wild-type and zyxin-null fibroblasts with the membrane-permeable drug jasplakinolide which stabilizes actin filaments and decreases the critical concentration of monomers required for polymerization (Bubb et al., 2000
). Jasplakinolide-treated wild-type fibroblasts developed robust actin filament arrays detected by phalloidin staining, but jasplakinolide-treated zyxin-null fibroblasts did not exhibit such robust stress fiber arrays (). To quantitate a stress fiber thickness index (SFTI), we used an “erosion” spatial filtering approach to calculate decay constants of brightness curves (; see Materials and methods). This analysis confirmed that there was no statistically significant difference in phalloidin-stained actin filaments between untreated wild-type and zyxin-null fibroblasts (). However, jasplakinolide-induced actin stress fibers in wild-type fibroblasts were thicker than the fibers in zyxin-null fibroblasts (). When GFP-zyxin was expressed in the zyxin-null cells, the ability of the cells to generate robust actin filament arrays in response to jasplakinolide was restored. Zyxin-null cells expressing GFP-zyxin () showed more robust actin stress fibers () after exposure to jasplakinolide than adjacent cells that were not expressing the transgene. Rescue of the zyxin-null cells' ability to build robust stress fibers by expression of GFP-zyxin was confirmed by quantitative SFTI analysis ().
Figure 9. Zyxin-null cells fail to augment actin stress fibers in response to jasplakinolide. Wild-type (A) and zyxin-null (B) fibroblasts were treated with vehicle or jasplakinolide and then fixed and stained by phalloidin. Jasplakinolide induced thick actin stress (more ...)
Dynamic regulation of zyxin localization during stress fiber remodeling
To begin to assess how zyxin might contribute to the cellular response to jasplakinolide, we examined the behavior of zyxin in jasplakinolide-treated cells. As can be seen in , zyxin underwent a dramatic redistribution in response to exposure of cells to jasplakinolide. Zyxin was lost from focal adhesions and accumulated along the actin stress fibers (). Under those conditions, zyxin was colocalized with caldesmon along actin filaments (). In untreated fibroblasts, zyxin was colocalized with vinculin at focal adhesions (). In contrast to zyxin, which left focal adhesions in response to jasplakinolide, vinculin was retained at focal adhesions (), illustrating the specificity of zyxin's redistribution and also illustrating that the focal adhesions were not disassembling in response to jasplakinolide. In contrast with vinculin, which was retained at focal adhesions in cells exposed to jasplakinolide, Ena/VASP proteins redistributed from focal adhesions to actin filaments concomitant with zyxin redistribution (). This VASP redistribution to actin stress fibers depended on the presence of zyxin (). VASP accumulation in lamellar extensions was zyxin independent ().
Figure 10. Jasplakinolide induces zyxin and VASP localization to actin filaments. Wild-type fibroblasts treated with jasplakinolide (JAS) or DMSO (control) were evaluated by indirect immunofluorescence microscopy. Zyxin localization in control fibroblast (A) and (more ...)