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1Instituto de Investigaciones en Ciencias de la Salud Humana, Universidad Nacional de La Rioja, La Rioja; and 2Cátedra de Biología Celular, Departamento de Ciencias Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, and 3Instituto de Química y Fisico-Química Biológicas-Consejo Nacional de Investigaciones Cientificas y Tecnicas, Buenos Aires, Argentina
Submitted 18 May 2007 ; accepted in final form 20 December 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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stress fibers; cell-matrix adhesion; vinculin; talin; actin filaments
Bradykinin (BK) is an endogenous intrarenal hormone that plays an important role as a modulator of the renal function, acting as a vasodilator and diuretic agent (29). In addition to its vasoactive actions, BK exerts significant effects on cellular growth and proliferation of cultured rat mesangial cells (1, 7). It is known that the signal that allows growth to proceed depends, in part, on the adhesion of cells to the extracellular matrix. Moreover, BK induces tyrosine phosphorylation of FA proteins such as focal adhesion kinase and paxillin (17–19). These findings prompted us to evaluate the potential role of BK in modulating FAs and the actin cytoskeleton in rat renal papilla collecting duct cells under physiological conditions.
Although FAs cannot be detected in histological preparations from intact tissue, they can be observed in cultured cells. Therefore, we took advantage of the fact that primary cultured papillary collecting duct cells preserve their tendency to interact with their self-formed extracellular matrix, mimicking their behavior in intact tissue. By using such experimental strategy, we demonstrated that BK induces a restructuring of FAs and remodeling of stress fibers in renal papillary collecting duct cells. By modifying the plasticity of cell adhesion structures, BK could be affecting collecting duct cell morphology and function.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Cell cultures and BK treatment. Primary cultures of papillary collecting duct cells were performed according to Stokes et al. (27). Briefly, renal papillae were minced to 1- to 2-mm3 pieces and incubated at 37°C in sterile TBS containing 0.1% collagenase II (Sigma, St. Louis, MO) under 95% O2-5% CO2. After 40 min, digestion was stopped and isolated cells and structures were separated by centrifuging at 175 g for 10 min. The "crude-pellet" containing most papillary cell types, tubular structures, and tissue debris was washed twice and resuspended in DMEM with F12 (1:1, GIBCO, Invitrogen), 10% FBS (Natocor, Córdoba, Argentina), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO, Invitrogen). The "enriched collecting duct pellets" were obtained by centrifugation at 60 g for 1 min and resuspended in an adequate volume of DMEM/F12. Depending on the methodological requirements, enriched tubular suspensions were seeded in 100-mm dishes or in sterile dry-glass coverslips placed in six-well multidishes. After growing at 37°C for 96 h, cultures were treated with 1 µM BK (Sigma) for 1, 5, and 10 min. Incubations were stopped on ice, and cells were rapidly processed for microscopy or cellular fractionation. When experiments were performed in the presence of the PLC inhibitor U73122 [GenBank] (Sigma), cultures were preincubated for 5 min before BK stimulation.
Immunofluorescence staining. Cultures treated as described above were washed with PBS and fixed with methanol (at –20°C for 10 min) and acetone (at –20°C for 4 min), and blocked with 3% goat normal serum (Vector Laboratories) in PBS. After fixation, cells were stained with either mouse anti-vinculin (Sigma) or mouse anti-talin (Sigma) overnight at 4°C in goat serum containing PBS. Detection of primary antibodies was performed with FITC-conjugated F(ab')2 fragment goat anti-mouse IgG (Jackson ImmunoResearch) secondary antibody. In PLC-β inhibitor U73122 [GenBank] and cytoskeleton studies, cells were washed with PBS and fixed with 4% paraformaldehyde solution in PBS for 15 min, permeabilized with a 0.1% Triton X-100 in PBS for 15 min, and blocked with 1% bovine serum albumin (Sigma) in PBS. The cells were then washed with PBS and stained with either mouse anti vinculin (Sigma) or mouse anti-talin (Sigma) for 1 h. After being washed with PBS, the cells were stained with the tetramethylrhodamine isothiocyanate-conjugated F(ab')2 fragment goat anti-mouse IgG (Jackson ImmunoResearch), and with FITC-coupled phalloidin (Sigma). Finally, the cells were mounted using Vectashield Mounting Media (Vector Lab) and stored at 4°C until analysis. Specimens were examined with an Olympus FV300 Confocal Microscope (model BX61), with FluoView version 3.3 acquisition software provided by the manufacturer.
Image analysis. The confocal images were analyzed using Image-Pro Plus version 5.1.2 (Media Cybernetics). All adhesion sites (FAs) in 5–10 randomly selected cells were analyzed for generation of quantitative data sets for each treatment, and three independent experiments were performed. Cells were examined with a x100 Plan Apo oil objective (numerical aperture 1.4), resulting in a pixel length of 0.1388 µm after performance of spatial calibration. To even out background variations in our immunofluorescence images, we applied a "flatten filter". This is often done to prepare an image for count/size operations because flattening reduces the intensity variations in the background pixels. To select the intensity range of the objects (FAs) to be counted and measured, we applied the software command to perform the "segmentation" of the image. This application segments the image into objects and background. The total number of vinculin- and talin-immunostained FAs per cell was counted. To this end, the contour of each cell in the confocal image (area of interest) was manually drawn by using the mouse pointer of the program. For each matrix-adhesion site, the following morphometric parameters were calculated by the software: size (area of FA plaque, in µm2), average length (AL; caliper of FA plaque, in µm), and the axial ratio (AR) of the best-fitted ellipse, which defines FA shape (ratio of the length of the long and short axes). A value of AR <7 corresponds to a rounded-to-ellipsoid adhesion, and a value of AR >7 corresponds to an elongated structure (36). We established a valid range for each measurement using the set-range command of the software, specifying a minimum value for each measurement: 0.25 µm2 for the size, 0.2 µm for average length, 0.2 µm for the minor axis, and 0.5 µm for the major axis.
Cellular fractionation. As described above, cells were grown on 100-mm dishes, treated with BK, and rapidly scraped off with a rubber policeman and collected in buffer lysis solution (phosphate buffer, pH 7.4, containing 0.15% NaCl and 0.03% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin). To ensure the complete disruption of the cells, suspensions were passed 10 times through a 29-gauge needle and, after that, homogenized with a Teflon pestle in a 1-ml Potter cup. This procedure was repeated twice. The resulting homogenate was layered on a double volume of 25 mM Tris·HCl, pH 7.4, at 4°C, buffer solution containing 0.50 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, and centrifuged at 900 g for 15 min. The pellet was washed twice and used as the nuclear fraction. Postnuclear supernatants were pooled and centrifuged at 105,000 g. The resulting pellet and supernatant were used as microsomal and cytosolic fractions. Aliquots from total cell lysates, nuclear, microsomal, and cytosolic fractions were assayed for protein content by the method of Lowry. To study the purity of the subcellular fractions, we determined the absence or the presence of Na+-K+-ATPase β1 in cytosolic, microsomal, and nuclear fractions by Western blotting analysis (Supplemental Fig. S1; all supplemental material in the article is available in the online version of this article on the journal web site).
Western blot analysis. Aliquots containing 40 µg of proteins of cell lysates, nuclear, microsomal, and cytosolic fractions were boiled for 5 min with Laemmli buffer, resolved by electrophoresis in a 7.5% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membrane. After blotting, membranes were treated with 5% nonfat milk in TBS-Tween 20 and incubated with mouse anti-vinculin (Sigma), mouse anti-talin (Sigma), and mouse anti-Na+-K+-ATPase β1 (Santa Cruz Biotechnology). Primary interaction was evidenced by using the enhanced chemiluminiscence kit (Amersham Biosciences) or avidin-biotin-peroxidase (Dako) and 3,3'diaminobenzidine (Sigma). Membranes corresponding to cell lysates and cytosolic fractions were stripped and retested with anti-actin antibody to verify that equivalent amounts of protein were loaded from the samples. To control the protein loading of microsomal and nuclear fractions, membranes were stained with Ponceau S or Coomassie blue. The intensity of each band was estimated by optical densitometry with Gel-Pro Analyzer 3.1.
Statistics. Results are expressed as means ± SE. Data from control and different treatments were analyzed by ANOVA, and significant differences were assessed by an a posteriori Dunnett multiple comparisons test or t-test (P < 0.05).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3A, vinculin- and talin-stained FAs appeared as bright and elongated structures. After 1 min of BK treatment, most of the vinculin-stained FAs were dissipated and vinculin was localized in the perinuclear region (Fig. 1B). The quantitative analysis showed that only 25% of the vinculin-stained FAs remained assembled (Fig. 2A). After 5 min, the number of FAs was still low (25% of the control value), and after 10 min it appeared that vinculin-stained FAs began to reassemble, since the number of FAs per cell was recovered, rising to 65% of the control value (Figs. 1, C and D, and 2A).
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Image analysis has proved to be a useful tool to investigate cell-matrix adhesion structures under different physiological stimuli (30, 36). Therefore, to analyze the effect of BK treatment on FAs in more detail, we quantified various morphometric parameters of FAs including size (area of FA plaque, in µm2), average length (caliper, in µm), and the AR, which defines FA shape (ratio of the length of the long and the short axes of the best-fitted ellipse). Quantization of FA morphological parameters were calculated by the image-analysis program and performed as described in MATERIALS AND METHODS. The vinculin-stained FAs of untreated cultured rat renal papillary collecting duct cells were round to ellipsoid (AR <7), measuring 0.42–5.87 µm (minimum-maximum average length value, respectively) with a size of 6.94 ± 0.84 µm2 (Fig. 2, B–D). Treatment of cultured cells with BK induced a decrease in vinculin-stained FA average length, which was completely reversed by the inhibition of PLCβ (Fig. 2B). As shown in Fig. 2C, the axial ratio of vinculin-stained FAs did not change after BK treatment, denoting that the shape of the vinculin-stained FAs was not affected by BK, thus preserving their rounded-to ellipsoid shape (AR <7). The size of vinculin-stained FAs was substantially reduced (Fig. 2D). After 1 min of BK stimulation, the size was reduced in 
35% of their initial size, and no recovery was observed after 10 min. Such a BK-induced decrease in size was completely abolished by the previous treatment with the PLCβ inhibitor U73122
[GenBank]
(Fig. 2D). Neither the morphometric parameters nor the number of FAs presented significant effects when control cells were preincubated with the PLCβ inhibitor (Fig. 2, A–D), which reflects that basal, nonstimulated PLCβ activity was not involved in the dynamics of vinculin-stained FAs.
Figure 3 shows representative images of the experiments where the effect of BK in talin-stained FAs was studied. BK did not induce dissipation of talin-stained FAs at any time studied, as occurs with vinculin-stained FAs (Fig. 3, A–D). To avoid selection bias, we performed a quantitative analysis to investigate the number of talin-stained FAs per cell and we found a significant increase after 5 min of BK treatment (Fig. 3, C and E). The pretreatment of collecting duct cultured cells with the PLCβ inhibitor U73122 [GenBank] before BK stimulation abolished the increase in talin-stained FAs resulting from 5 min of BK treatment (data not shown).
Thereafter, we also searched for BK-induced morphometric changes in talin-stained FAs. The average length did not present significant changes, while the size tended to increase although not statistically significantly (Fig. 3F). However, BK induced the appearance of some >8-µm-long FAs (Fig. 3C, arrowhead) not observed in untreated cells. No changes in the shape of talin-stained FAs were observed, as reflected in the values of axial ratio (Fig. 3H).
BK affects the amount and intracellular distribution of FA proteins vinculin and talin. To evaluate whether BK provokes changes in the amount and/or localization of the FA proteins vinculin and talin, we performed a Western blot analysis in total homogenates and intracellular preparations of collecting duct cultured cells either untreated or treated with BK. As seen in Fig. 4A, after 1 min of BK stimulation the amount of vinculin in the total cell lysate decreased, and thereafter increased. To evaluate the intracellular distribution of vinculin, membrane, cytosolic, and nuclear preparations were obtained from primary cultured collecting duct cells either untreated or treated for 1, 5, and 10 min with BK. The results showed that in control preparations vinculin was present in both cytosolic, membrane, and nuclear fractions (Fig. 4A). BK stimulation induced an increase in cytosolic vinculin with some oscillation at 5 min (Fig. 4A). In microsomes, a decrease in vinculin signal from 1 to 5 min of stimulation, and an increase thereafter were observed (Fig. 4A). These results are in agreement with the immunofluorescence images, which showed that the number of vinculin-stained FAs per cell decreased reversibly after BK treatment, concomitantly with an increase in fluorescence in the cytosol (Figs. 1, A–D, and 4A). In nuclear preparations from control cells, a high vinculin signal, which progressively decreased after BK stimulation, was observed (Fig. 4A).
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BK affects actin cytoskeleton organization by a PLC-mediated mechanism. Figures 5, 6, and 7 show representative images where the effect of BK in actin cytoskeleton organization was studied in two separate sets of experiments. In one set we analyzed the stress fibers associated with vinculin, and in the other set we analyzed the talin-associated stress fibers.
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In summary, in untreated cells, short SFs predominated and were located in the center of the cell (vinculin- and talin-associated ones), while in BK-treated cells, BK induced the formation of long SFs mainly located in the periphery of the cell (neither vinculin- nor talin-associated ones).
After BK stimulation, cultured cells maintained the flattened shape and remained attached during all the experimental time. However, cell contraction was observed, which was more evident after 5 min of BK stimulation (Fig. 5, C and G*). In addition, BK also promoted the formation of filopodia, more evident after 5 min of BK stimulation, which appeared connecting the cells to each other (Figs. 5G*, 1 and 2, arrowheads, and 6G, arrow).
The pretreatment of cultured cells with the PLCβ inhibitor U73122 [GenBank] prevented the loss of ventral SFs associated with vinculin-stained FAs, but not the appearance of transversal arc after 1 and 5 min of BK stimulation (Fig. 7, B, C, F, and G, arrowheads). Conversely, the pretreatment with the inhibitor abolished all the changes observed in the actin cytoskeleton after 10 min of stimulation. The appearance of a great number of vinculin-associated ventral SFs denotes the reversibility of BK's effect on the actin cytoskeleton (Fig. 7H, arrowhead). Moreover, the characteristic BK-induced cell contraction was not observed, and the cells looked like the untreated ones (Fig. 7, D and H).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FAs are hierarchical multiprotein structures formed by sequential binding of several FA proteins (34), which through the assembling and dissembling of FA proteins, constitute the remodeling process that gives plasticity to the cell. In this process, talin directly binds integrin, thus initiating the formation of the FAs by recruiting vinculin (34). In the present report, we show that BK induces the dissipation of vinculin-stained FAs but stabilizes FAs that are talin-stained, thus assuring the cell-extracellular matrix attachment. These results could be of physiological relevance. Since FAs are the most stable points of the cell tethering to the extracellular matrix, their dissipation is expected to provoke the detachment of the cells. In the specific case of collecting duct cells, the preservation of the cell-extracellular matrix attachment is a condition of survival, since it is known that cell-extracellular matrix detachment provokes cell death by anoikis (31). Considering that collecting duct cells constitute a target for BK, the fact that BK does not evoke total FA dissipation is consistent with its physiological role as a modulator of tubular function and not as a potential deleterious agent. An interesting detail in the modulatory effect of BK is the induction of some longer talin-stained FAs, known as supermature FAs, which are efficient in promoting tissue contraction in culture myofibroblasts by providing high adhesion to the extracellular matrix (11). As we show in this work, BK indeed provokes cell retraction, and such supermature FAs, although not abundant, may act as contributors to such a process. We consider that the fact that, parallel to the decrease in the number, average length, and size of vinculin-stained FAs, BK evokes an opposite effect on talin-stained FAs, appearing as a compensatory effect of BK to fulfill its physiological nondeleterious role in collecting duct cells.
Stress fibers are contractile actomyosin bundles, which are essential for cell adhesion to the substratum and for changes in cell morphology (14, 25). In the current work, we demonstrate that untreated cultured collecting duct cells possess all the different types of SFs described in migrating cells (14, 25). It has been reported that ventral SFs play an important role in cell adhesion (14). In untreated cultured cells, we showed a preponderance of ventral SFs, which correlates well with the characteristics of collecting duct cells, which need a strong adhesion to the extracellular matrix to maintain their typical tubular organization and function. It has been demonstrated, in a human osteosarcoma cell line (U2OS cells), that the three distinct types of stress fibers interact directly with each other and form a continuous and dynamic actin filament network (14). Dorsal SFs and transverse arcs are generated by the novo polymerization of the actin filament or by condensation of smaller filament fragments, whereas ventral SFs can be generated through reorganization of preexisting dorsal SFs-transverse arc network (14). Although many proteins have been implicated in the in vivo process of actin filament assembly, the cellular or extracellular factors that induce their interconversion have not yet been described. Here, we show that BK induces the reversible interconversion of the various types of SFs reflected by the rapid decrease in ventral SFs associated with vinculin, concomitant with the appearance of transverse arcs. At a longer time (10 min), dorsal SFs appear to act as precursors of newly formed ventral SFs, restoring the initial state. Previous work performed using subconfluent, serum-starved Swiss 3T3 cells have shown that BK induces loss of SFs (16). In this study, we observed that BK induces a reversible remodeling of the actin filament assemblies rather than a dissipation of them, and we propose that BK is an endogenous modulator of the actin filament network in collecting duct cells.
Despite the remodeling of the cytoskeleton induced by BK, the cells maintained a flattened shape and remained attached to the growth surface. We consider that the preservation of the talin-stained FAs, which in turn permits the persistence of the talin-associated ventral SFs, is the primary factor for the maintenance of collecting duct cells attached to the surface. We are tempted to suggest that the resistance of talin to be mobilized by BK is probably an important difference between collecting duct cells and migratory cells, which, without doubt, need the complete dissipation of FAs to perform migration.
The biochemical analysis of the cytoskeletal proteins vinculin and talin demonstrated that BK affected their amount and subcellular distribution differently. The results suggest that BK stimulation induced an intracellular redistribution of vinculin rather than changes in the total amount of the protein. Interestingly, the vinculin signal was highly positive in nuclear preparations of control cells. It has been reported that vinculin can be associated with β-catenin and recruited into the nucleus in Madin-Darby canine kidney cells overexpressing β-catenin, but the physiological role of this observation has not yet been determined (24). The fact that BK induced a rapid and progressive decrease in the nuclear content of vinculin, together with its high basal content, leads us to suggest that such a pool of vinculin can be not only the main source of the early increase in cytosolic vinculin but also the source of the microsomal replenishment observed thereafter. Taking into account that our study was performed with cells not genetically manipulated, our results could be providing new experimental evidence supporting a novel role of the vinculin nuclear pool, which can be mobilized by BK under physiological conditions.
In the case of talin, the biochemical experiments revealed that BK caused a sustained increase in the protein in microsomal preparations, which is consistent with the persistence of the talin-stained FAs observed after BK stimulation. The further increase in the cytosolic level of talin and the absence of any potential source of talin can indicate that BK induced an increase in protein concentration rather than an intracellular redistribution. On the other hand, the rapid increase in the level of talin suggests that such an increase was due to an inhibition of degradation rather than to an increased synthesis. We have no direct evidence to demonstrate this hypothesis. However, it is known that FAs are dynamic multiprotein complexes, and it has been recently demonstrated that the Ca2+-dependent cystein protease calpain is directly involved in the FA turnover (2) and that various cytoskeletal proteins, including talin but not vinculin, are targets for calpain-mediated degradation (13). Calpain is present in the cytosol but translocates to the membrane for activation. It is accepted that its binding to membrane phospholipids lowers the requirement of Ca2+ concentrations for activation. On the other hand, calpain is physiologically controlled by the endogenous inhibitor calpostatin, whose binding to calpain requires Ca2+ concentrations similar to those required for the in vitro activation of calpain (5). It is known that the signal transduction initiated by the activation of PLCβ produces an increase in intracellular Ca2+ concentrations. We consider that such elevation of Ca2+ can favor the calpostatin-calpain binding, thus inhibiting calpain activity. As talin is a target for active calpain, its inhibition can result in an increase in talin concentration. On the other hand, since BK is a physiological activator, the termination of the signal initiated by the BK-mediated PLCβ activation could release calpain from calpostatin binding, thus restoring its activity. This consideration could explain the decrease in talin amount observed in the total lysate as well as in the cytosolic preparation after 10 min of BK stimulation. Our hypothesis can be supported by a recent report that demonstrates that inhibition of calpain activity or overexpression of calpostatin stabilizes FA structures (2).
We show here that BK induces reversible cell retraction with the appearance of multiple thin filopodia. The appearance of supermature talin-stained FAs, together with the great increase in transversal arcs, both involved in the induction of cell contraction, can explain the cell retraction observed after BK stimulation. As compensation, long thin filopodia seem to connect cells to each other, probably favoring the complete reversibility of the BK effect.
We have previously reported that activation of phosphoinositide-specific PLCβ is an early event in BK stimulation of rat renal papillary tissue (26). PLCβ initiates a signaling pathway by hydrolysis of the acidic phospholipid phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. On the other hand, PI(4,5)P2 acts as a regulator of actin dynamics and FA turnover (3, 28), and many cytoskeletal proteins involved in cell adhesion, including vinculin and talin, bind PI(4,5)P2 (9, 20, 23). Both FA activation and integrin binding to talin are dependent on the local PI(4,5)P2 availability (9, 20). Since BK stimulates PI(4,5)P2 hydrolysis by PLC activation, we attempted to determine whether BK can modulate FA restructuring by this mechanism. We show here that the pretreatment of primary cultures of rat renal papillary collecting duct cells with the PLC inhibitor U73122 [GenBank] partially abolished the BK-induced changes in cytoskeleton organization. In agreement with the present results, it has been shown that stimulation of National Institutes of Health 3T3 cells with platelet-activating factor (PAF), EGF, or PDGF reduces adhesion energy, whereas addition of the PLC inhibitor U73122 [GenBank] prevents this reduction, suggesting that PI(4,5)P2 works as a second messenger, regulating the adhesion energy between the cytoskeleton and the plasma membrane (22). On the other hand, local changes in PI(4,5)P2 concentration can regulate cortical plasma membrane-cytoskeleton structure by directly altering interactions between PI(4,5)P2 and cytoskeletal anchoring proteins (22, 28). The question is why BK dissipates vinculin but not talin from FAs. We have no direct evidence for an explanation. However, it has been reported that talin has high affinity for phosphatidylinositol-4-phosphate (PI4P) (20), whose concentration in plasma membrane is higher than PI(4,5)P2. On the other hand, we performed the analysis of an immunoprecipitation with anti-vinculin or anti-talin and found that while talin was mostly bound to PI4P, the phosphoinositide that coprecipitated with vinculin was PI(4,5)P2 (N. Sterin-Speziale, unpublished observations). Thus it is expected that after BK-induced degradation of PI(4,5)P2, talin can still remain bound to PI4P, thus forming part of the FAs.
In conclusion, the present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLC. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 and the bradykinin B2 receptor—importance of growth conditions. Biochem Biophys Res Commun 326: 894–900, 2005.[CrossRef][Medline]
-smooth muscle actin to stress fibers. J Cell Biol 172: 259–268, 2006.This article has been cited by other articles:
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  M. G. Marquez, M. d. C. Fernandez-Tome, N. O. Favale, L. G. Pescio, and N. B. Sterin-Speziale Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1181 - F1191. [Abstract] [Full Text] [PDF]
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