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Vol. 18, Issue 3, 827-838, March 2007
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*Laboratoire de Morphogenèse et Signalisation Cellulaires, Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique/Institut Curie, 75248 Paris Cedex 05, France; Laboratoire Physico-Chimie Curie, Unité Mixte de Recherche 168, Institut Curie/Centre National de la Recherche Scientifique/Universités Paris 6 and 7, 75231 Paris Cedex 05, France; and Laboratoire de Physiologie Cellulaire Végétale, Unité Mixte de Recherche 5168, Commissariat à l'Energie Atomique/Centre National de la Recherche Scientifique/Université Joseph Fourier, 38054 Grenoble Cedex 9, France
Submitted May 17, 2006;
Revised December 4, 2006;
Accepted December 7, 2006
Monitoring Editor: Yu-li Wang
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Matsudaira and Burgess, 1979; Robine et al., 1985). Microvilli are thin membrane extensions at the apical pole of epithelial cells, they largely increase the cell surface available for absorption and secretion. Microvilli have been extensively studied and shown to be a good model to understand morphogenesis and differentiation of cells. They are sustained by bundles of parallel actin filaments. Numerous actin-binding proteins among which is villin are responsible for the organization and the maintenance of the actin cytoskeleton in microvilli (Revenu et al., 2004).
Villin belongs to the gelsolin family. All proteins of this family are organized around a conserved module of 
120 amino acids. This module is repeated three times in the proteins severin and fragmin that belong to lower eukaryotes (Dictyostelium and Physarum, respectively). Along evolution, a further duplication of the three modules occurred in gelsolin and villin, which contain six modules (Arpin et al., 1988; Bazari et al., 1988). These six modules named V1–V6 constitute the core of the villin protein (see Figure 1). Villin contains an additional C-terminal domain that is absent in gelsolin, called the headpiece (Arpin et al., 1988). The villin effects on actin have been analyzed in vitro and were shown to depend on the calcium concentration (Bretscher and Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al., 1980). At low calcium levels (10–100 nM), villin bundles actin filaments. At micromolar calcium concentrations, villin starts to nucleate and cap the barbed ends of actin filaments. At higher calcium concentrations, villin is able to sever actin filaments, and this activity reaches its maximum efficiency above 100 µM calcium. Phosphorylation has also been shown to modulate the properties of villin. The in vitro phosphorylation of different tyrosines in villin enhances its severing and inhibits its bundling activities (Zhai et al., 2001, 2002). The domain responsible for the capping and severing activities of villin is included in the V1 and V2 modules where an F-actin binding site has been localized (Matsudaira et al., 1985; Janmey and Matsudaira, 1988). The second half of the villin core (V4–V6) is essential for its nucleation property (Friederich et al., 1999). The ability to bundle filaments is due to the presence of an additional F-actin–binding site in the headpiece (Glenney and Weber, 1981).
Because villin is able to bundle actin filaments, it was thought to be involved in the formation of microvilli. Indeed, the overexpression of villin in fibroblastic-like cells CV1 is able to induce the formation of microvilli (Friederich et al., 1989), and the transcriptional silencing of villin in Caco-2 cells affects their distribution and length (Costa de Beauregard et al., 1995). These data obtained in cell cultures supported a role for villin in the formation and maintenance of microvilli. However, the knock out of the villin gene in mice (vil–/–) did not induce any detectable ultrastructural alteration of the microvilli of intestinal epithelial cells (Ferrary et al., 1999). Nevertheless, the intestinal epithelium of these mice exhibited a decreased cellular response to stresses and to the induction of lesions by dextran sodium sulfate, treatments that result in intracellular calcium rise (Ferrary et al., 1999). After lesions, enterocytes have to undergo an epithelio-mesenchymal transition (EMT) to reconstitute the epithelial barrier. The studies on the vil–/– mice suggested that the remodeling of the actin cytoskeleton needed in EMT was impaired in the epithelial cells knocked out for villin. In support of this hypothesis, the efficiency of cell motility and morphogenesis (Athman et al., 2003) as well as the infectious process of Shigella flexneri (Athman et al., 2005) were reported to be increased in villin expressing cells, confirming a new role for villin in actin cytoskeleton dynamics.
Our hypothesis is that the villin-severing activity is responsible for the enhancement of actin dynamics. By cutting actin filaments, villin would increase the number of free barbed and pointed ends and enhance the cycling of actin needed for actin based motility. To test this hypothesis, we designed a severing mutant of villin. We demonstrate that this mutant of villin has a strongly reduced severing activity while retaining the other known activities. Because only the severing activity of villin is affected, we used this mutant in vivo and in an in vitro motility assay to analyze the impact of villin severing on actin-based motility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was used as template for the mutation of putative sites responsible for villin-severing activity. All the mutations were introduced using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Complementary primers containing the desired mutations were designed as described previously (Kumar et al., 2004a) for the mutations D61N, D86L, and A93G. For the RRI variant (86DDFLKG/NTLLKE, 125GMKHV/AMHKTS), the two regions and the insertion were introduced as follows. The primer sense ACC ACA CAG ATG AAT ACC TTG CTG AAG GAG CGG GCT GTG CAG CAC and the antisense GTG CTG CAC AGC CCG CTC CTT CAG CAA GGT ATT CAT CTG TGT GGT were used to mutate the amino acid sequence 86DDFLKG in NTLLKE. The primer sense GGG GGC GTG GCT TCT GCC ATG AAG ACC TCG GAG ACC AAC TCC and its complementary sequence GGA GTT GGT CTC CGA GGT CTT CAT GGC AGA AGC CAC GCC CCC were used to transform the amino acid sequence 125GMKHV in AMKTS. The primers sense GTG GCT TCT GCC ATG CAC AAG ACC TCG GAG ACC and antisense GGT CTC CGA GGT CTT GTG CAT GGC AGA AGC CAC were used to introduce an histidine in position 127. The whole gene was sequenced to ensure that only the desired mutations were introduced.
Wild-type (WT) villin and its variants were produced using standard procedures (Friederich et al., 1999). Briefly, villin and its variants, tagged with glutathione S-transferase (GST), were purified from the soluble fraction of Escherichia coli Bl21 bacteria using glutathione-Sepharose beads. The beads bound to GST-villin were digested with thrombin. Thrombin was then removed from the eluates containing villin and variants by an incubation with p-amino-benzamidine agarose beads (Sigma, St. Louis, MO). Proteins were dialyzed overnight against phosphate-buffered saline (PBS). They were then aliquoted, snap-frozen in liquid nitrogen, and stored at –80°C.
Actin Polymerization and Depolymerization Assays Using Fluorescence Spectroscopy
Villin activities toward actin filaments were tested by the use of N-(1-pyrene)iodoacetamide–labeled actin (pyrene-actin). Monomeric pyrene-actin fluoresces weakly at 388 nm when excited at 365 nm. When polymerized, a strong fluorescence peak appears at this wavelength. Hence, if actin polymerizes, the fluorescence intensity increases and vice versa (Kouyama and Mihashi, 1981).
Actin was purified from rabbit skeletal muscle acetone powder according to the procedure from Spudich and Watt (1971) and kept in G-buffer (2 mM Tris, 0.2 mM CaCl2, 0.2 mM ATP, 1 mM dithiothreitol [DTT], pH 8.5) at –80°C. The labeling of actin with pyrene-iodoacetamide was obtained and quantified following standard procedures (Kouyama and Mihashi, 1981). Fluorescence measurements were performed at room temperature using a fluorescence spectroscope (Perkin-Elmer, Wellesley, MA) driven by the Flwinlab software. The excitation wavelength was set at 365 nm and the emission wavelength at 388 nm. All measurements were done on samples of 400 µl.
Severing of Actin Filaments
Actin (2 µM, 30% labeled) was polymerized overnight at 4°C in F-buffer (2 mM Tris, pH 7.2, 100 mM KCl, 2 mM MgCl2, 0.2 mM ATP, 1 mM DTT). At t = 0, F-actin was diluted to 100 nM by the addition of F-buffer containing CaCl2, villin, and its variants at specified concentrations. The fluorescence intensity was recorded over 330 s. The severing activity was estimated as described previously (Gettemans et al., 1995; Friederich et al., 1999). The decrease in fluorescence per minute of the linear range of the curves was considered as corresponding to 100% activity for WT villin and 0% activity for control experiments without severing protein.
Nucleation of Actin Polymerization
G-actin (2 µM, 10% labeled) was preincubated for 10 min on ice with 24 nM of villin or its variants in G-buffer containing 20 µM CaCl2. At t = 0, polymerization was induced by the addition of KCl and MgCl2 to reach the final concentrations of 100 and 2 mM, respectively. The fluorescence intensity was recorded over 30 min.
Elongation Assay To Determine the Affinity of Villin for the Barbed Ends of Actin Filaments
Actin filaments (1 µM final) were incubated for 5 min at room temperature with various concentrations of WT or RRI villin in F buffer containing 2.5 µM free Ca2+. Elongation was initiated by the addition to the actin filament mixture of 1 µM actin monomers (5% pyrene-labeled) bound to 2 µM profilin (human profilin 1 purified as described previously; Fedorov et al., 1994). The polymerization was followed by changes in pyrene fluorescence using a Xenius SAFAS (Safas SA, Monaco). The affinity of the proteins for the barbed ends of actin filaments was determined by the variation of the initial rate of elongation as a function of the concentration of villin, using the following equation:
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Low-Speed Sedimentation Assay
Actin was polymerized in F buffer and then complemented to reach the final composition of the bundle buffer (5 mM KH2PO4, pH 7, 2 mM EGTA,1 mM DTT, 100 mM KCl, 1 mM MgCl2). F-actin (10 µM) was incubated for 1 h at room temperature with villin and its variants (2.5 µM) to allow bundling to occur. The proteins were centrifuged at 8000 x g for 15 min in order to pellet aggregates of filaments but not single filaments. The supernatant was separated from the pellet, and both were loaded on acrylamide gels. The gels were stained with Coomassie brilliant blue.
Electron Microscopy Analysis of Bundling Activity
To analyze bundling activity, F-actin was polymerized in F buffer and subsequently complemented to reach the composition of the bundle buffer. F-actin (3 µM) was incubated overnight at 4°C in the presence of 1.5 µM villin WT and variants. Actin filaments were incubated on carbon-coated grids in the presence of 2 mM phalloidin to stabilize filaments. They were fixed with 1% glutaraldehyde and negatively stained by incubation with 2% uranyl acetate. The observations were made with a Philips CM120 electron microscope (FEI, Eindoven, The Netherlands).
Expression of Villin and Variants in Mammalian Cells
Villin WT and mutants cDNA were cloned in the vector pEYFP-C1 (Clontech, Palo Alto, CA) within the restriction sites EcoRI and SalI. CV1 and LLCPK-1 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) complemented with 10% fetal calf serum. CV1 cells were transfected by nucleofection (Amaxa, Gaithersburg, MD) according to the optimized procedure (solution V, program A33). LLCPK-1 cells were electroporated. At day 5 after transfection, cells were fixed in PFA and extracted with 0.5% Triton X-100. Actin filaments were stained using TRITC phalloidin. Cells were examined under an upright motorized microscope (Leica DMRA2, Wetzlar, Germany) equipped with an interlined CCD camera (Roper CoolSnap HQ, Tucson, AZ) and a piezo-electric motor (LVDT, Physik Instrument, Karlsruhe, Germany) mounted underneath the objective lens for rapid optical Z sectioning. The system was controlled by Metamorph software (Molecular Devices, Downingtown, PA). Stacks of images, taken with a 0.2-µm plane-to-plane distance, were restored using the Fast Iterative Constrained PSF-based algorithm (Sibarita, 2005).
For infection experiments, the yellow fluorescent protein (YFP) tag was replaced by mCherry (Shaner et al., 2004) using the restriction sites AgeI-BsrGI. Madin-Darby canine kidney (MDCK)-II cells were cultured as described above for LLCPK-1. The day before infection, MDCK-II cells were transfected with pmCherry-villin by nucleofection (Amaxa, solution L, program A24).
Infection by S. flexneri
The WT invasive strain of S. flexneri, M90T, and its variant expressing the green fluorescent protein (GFP; Rathman et al., 2000) were grown overnight in trypticase soy broth (TCS, Bio-Rad, Hercules, CA). On the day of infection, bacteria were subcultured to midexponential phase. MDCK cells transiently expressing mCherry-tagged WT or RRI villin were washed two times with PBS. S. flexneri was added at a multiplicity of infection of 200:1 in DMEM without serum and spun onto the cells at 2000 x g for 5 min at 20°C. The infected cells were then incubated at 37°C, 5% CO2. After 40 min, they were washed with DMEM containing 50 µg/ml gentamicin.
For immunofluorescence, WT M90T-infected cells were further incubated for 80 min. After an extensive wash with PBS, they were fixed in PFA and extracted with 0.5% Triton X-100. Actin filaments were stained with Alexa Fluor 488-phalloidin (Molecular Probes, Eugene, OR), and DNA was labeled with DAPI. The acquisitions were made with an epifluorescence microscope (Leica DM 6000B) coupled to a CCD camera (Roper CoolSnap HQ) and driven by the software Metamorph.
For videomicroscopy, GFP M90T infected cells were further incubated at 37°C for 20 min. 3D plus time sequences were acquired on an inverted microscope (Leica DMIRB2) with a set-up equivalent to the one described previously for rapid optical Z sectioning. The microscope was equipped with a thermoregulation system (Life Imaging Services, Reinach, Switzerland), allowing incubation of the cells at 37°C and 5% CO2. Illumination at 488 nm and 575 nm for GFP and mCherry, respectively, was performed with a monochromator. For each wavelength, stacks of four images were acquired every 5 s during 5 min, with a plane-to-plane distance of 1.5 µm. GFP bacteria-infecting cells expressing WT and RRI villin at equivalent levels or nontransfected cells were tracked with the Metamorph software. To evaluate the rate of disassembly, the average fluorescence intensity of a fixed area of the comet (diameter 5 pixels), located directly behind the bacterium in the initial frame analyzed, was monitored over 90 s.
In Vitro Actin-based Motility Assays
To reconstitute actin-based motility in vitro, polystyrene beads of 1 µm in diameter (Polysciences, Warrington, PA) were coated with the VCA domain of WASP as described previously and incubated in a motility medium (Bernheim-Groswasser et al., 2002; van der Gucht et al., 2005) containing 8 µM F-actin, 0.1 µM Arp2/3, 1 µM profilin, 5 µM ADF, and 0.1 µM villin WT or RRI. All the purified proteins were purchased from Cytoskeleton (Denver, CO) except villin that was produced as described above. Comet growth and bead movements were followed in phase-contrast microscopy. The velocity of the propelled beads was measured with the Metamorph software. To analyze the depolymerization of the actin array present in the comet, 10% Alexa 488–labeled actin (Molecular Probes) was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or point mutations targeting potential regulatory residues (Zhai et al., 2002; Kumar et al., 2004a). We decided to use a rational biochemical approach to design mutations suppressing the severing activity of villin. We took advantage of the work of F. S. Southwick, where he succeeded to introduce a severing activity to the capping protein CapG (Southwick, 1995). CapG is, as villin, a member of the gelsolin family but fails to sever actin filaments. The comparison of the primary structures of CapG with the severing proteins of the family allowed the identification of two sets of divergent amino acids in the severing domain. These amino acids, once mutated, were able to confer a severing activity to CapG (Southwick, 1995). We took the reverse strategy and changed the same amino acids in villin to the CapG sequence. This lead to the introduction of four mutations in two regions (R1 and R2) and to the insertion (I1) of one histidine in the second region (Figure 1). To simplify the nomenclature, we will refer to this mutant as RRI.
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; Burtnick et al., 2004). The corresponding R1 region in gelsolin is located at the C-terminal end of an 
-helix in G1 (Supplementary Figure 1). On activation, this site appears to be precisely situated at the binding site of a calcium ion that is shared between G1 and actin (Choe et al., 2002; McGough et al., 2003). The binding of this shared calcium ion is thought to directly mediate the binding of gelsolin G1 to actin. This suggests that the R1 mutations introduced in RRI villin and present in CapG could reduce the efficiency of G1 to bind calcium. The corresponding R2 region is part of the linker between domains G1 and G2. It makes intimate contact with actin in the G1–G2:actin complex (Burtnick et al., 2004). To undergo the conformational changes from the inactive calcium-free structure of gelsolin G1–G3 to the calcium-activated actin-bound one, the G1–G2 linker has to stretch out along actin. This allows the G1 domain to move away from G2 and to be properly directed toward the region where it will disrupt an actin–actin interaction. Hence the two mutated regions in RRI villin are part of strategic domains in the last step of the activation process of gelsolin severing. The RRI mutations could thus impair the activation and positioning of the V1 domain whose proper insertion in between two actin protomers leads to actin filament severing.
The RRI mutations are located upstream of the minimal F-actin–binding peptide (amino acids 133-147) mapped in villin V2 (de Arruda et al., 1992) and we can thus expect that the binding of villin to filamentous actin will not be impaired. Moreover, a gelsolin construct consisting of G2–G3 and part of the G3–G4 linker is efficient in capping actin filaments (Sun et al., 1994), and structural analysis support an active role of the G3–G4 linker in obscuring the barbed end of a capped filament (Burtnick et al., 2004). We can thus expect that the RRI mutations will not impair the other activities of villin, specifically capping and bundling.
The RRI Mutations Most Strongly Impair Villin-severing Activity
The severing efficiency of the RRI mutant has been analyzed and compared with other mutants that have been previously described to reduce the severing activity of villin (Kumar et al., 2004a), namely D61N, D86L, and A93G and their four possible combinations (D61N, D86L; D61N, A93G; D86L, A93G; D61N, D86L, A93G). We also took into account the mutation E74L, which is described to strongly reduce both the severing activity as well as the capping ability of villin.
Severing activity was measured by analyzing the effect of villin and villin mutants on the depolymerization of actin filaments labeled with pyrene. The filaments were diluted below the critical concentration of the pointed ends (100 nM). This results in their depolymerization, which is analyzed by the decrease in fluorescence intensity. The rate of depolymerization is dependant on the number of pointed ends available. By cutting filaments, villin should increase the number of pointed ends and hence increase the depolymerization rate.
As presented in Figure 2A, a slow depolymerization is observed in the control experiment without villin, whereas the addition of WT villin induces a very fast depolymerization. In these conditions (60 nM villin and variants, 200 µM Ca2+) the three point mutations D61N, D86L, and A93G and their four combinations did not allow the detection of a significant difference with the severing activity of WT villin (only D61N is depicted for clarity). However, the mutations E74L and RRI reduced the severing activity of villin to 29 and 16%, respectively. The RRI mutations are thus the most efficiently reducing villin-severing activity. The dose–response analysis of the severing activity of RRI villin compared with WT villin (Figure 2B) confirms this very strong impairment.
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Capping and Nucleation Activities Are Not Affected
Next, we determined if the RRI mutations were uniquely affecting villin-severing property without affecting the capping and nucleation activities.
The barbed-end capping activity of villin is saturated at 2.5 µM Ca2+, whereas severing is not (Northrop et al., 1986). Actin filament elongation assays were performed in the presence of 2.5 µM Ca2+ in order to determine the affinity of WT and RRI villin for the barbed ends of actin filaments (Figure 3A). Seeds consisting of unlabeled actin filaments were incubated with various concentrations of WT or RRI villin before the addition of pyrene-labeled actin monomers bound to profilin. The increase in fluorescence observed corresponds to the elongation of pyrene-actin-profilin complexes at the barbed ends of the seeds, because profilin-actin does not bind to pointed ends (illustrated for RRI villin in Figure 3A, inset). The decrease in the initial rate of elongation as a function of the concentration of villin reflects the saturation of filament barbed ends by villin (Figure 3A). We determined Kd values of 0.8 nM for WT villin (Figure 3A, squares) and 1.8 nM for RRI villin (Figure 3A, circles) binding to actin filament barbed ends. This demonstrates that WT and RRI villin have equivalent capping activities in the nanomolar range. Moreover, this affinity is similar to the ones reported for CapG (Southwick, 1995) and gelsolin (Laine et al., 1998).
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) show a delay of 1–2 min followed by a slow increase of fluorescence. RRI behaves like WT villin, and the RRI mutations thus do not significantly impair villin nucleation activity.
RRI Villin Is Still Able To Bundle Filaments
In vitro, at low calcium concentrations (<100 nM), villin is able to cross-link actin filaments in parallel bundles. This bundling ability is abolished by a deletion of the seven last C-terminal amino acids of villin, villin 
7 (Friederich et al., 1992). The formation of elongated bundle-like filaments observed upon incubation of Alexa-labeled F-actin with villin WT or RRI in presence of EGTA was confirmed by a low-speed sedimentation assay of actin filaments incubated with villin and its severing and bundling mutants, RRI and 7. Most of the actin was pelleted with villin WT and RRI, whereas it remained in the supernatant in the absence of villin or with villin 7 (Supplementary Figure 3). To confirm that WT and RRI villin bundled actin filaments, they were processed for electron microscopy (Figure 4). In the presence of WT and RRI villin, straight actin filaments closely aligned in parallel bundles were observed. Actin filaments alone or incubated with the bundling mutant 7 appeared as single intermingled filaments with few disorganized aggregates but no bundles could be observed.
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). In LLCPK-1 cells, the overexpression of villin lengthens the existing microvilli (Arpin et al., 1994; Loomis et al., 2003). These morphogenic effects are abolished by the disruption of villin bundling activity (Friederich et al., 1992). In Figure 5, we illustrate the consequences of the overexpression in LLCPK-1 cells of YFP-tagged villin WT and its variants. As for WT villin, RRI villin-overexpressing cells have elongated microvilli on their apical surface. Actin filaments colocalize with villin WT and RRI in these structures. On the contrary, although the bundling mutant of villin, 7, is properly localized with actin in the microvilli, it does not induce detectable structural modifications. The expression of the YFP tag alone (control) does not change the morphology of the cells. The overexpression of villin and its variants in CV1 cells lead us to the same conclusions: villin WT and RRI but not 7 induced the appearance of microvilli on the dorsal face of the cells (data not shown). In conclusion, disrupting the severing activity in the RRI villin affects neither its localization in microvilli nor its morphogenic consequences.
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). MDCK cells were thus transiently transfected with mCherry-tagged WT and RRI villin and infected with S. flexneri. As S. flexneri enters the cell, it recruits the cell's cytoskeleton machinery to produce an actin tail. This comet allows the bacteria to move inside the cell (Bernardini et al., 1989). As previously described for WT villin (Athman et al., 2005), RRI villin is also incorporated in the comet where it colocalizes with actin (Figure 6A). The movement of GFP S. flexneri in these cells was analyzed by videomicroscopy (see Supplementary Video 1 available as supplementary data). All bacteria that show a directional movement in WT and RRI villin-transfected cells are propelled by a comet labeled with mCherry-villin. The velocities (Figure 6B) calculated from the movies give an average rate of 7.0 ± 4.3 µm/min in nontransfected cells (nControl = 92), 6.7 ± 4.0 µm/min in RRI villin expressing cells (nRRI = 61), and 10.3 ± 3.7 µm/min in WT villin expressing cells (nWT = 80). The increase in velocity reported for the bacteria in the presence of WT villin is highly significant (Wilcoxon test, p = 2.213E-07; Athman et al., 2005) and is lost in the presence of RRI villin (Figure 6B). Indeed, the velocities of bacteria moving in nontransfected cells are not significantly different from those in RRI villin expressing cells (Wilcoxon test, p = 0.7813). The fluorescence decay of the comets labeled with villin was not significantly different in the presence of WT or RRI villin, indicating that the rate of disassembly is not modified, whereas the comets were significantly longer in the presence of WT as compared with RRI villin (4.0 ± 1.8 and 2.7 ± 1.7 µm, respectively, nWT = 39 and nRRI = 26, t test, p < 0.01). The wide distribution of the velocities as well as the heterogeneity in the length and intensity of the comets analyzed limit the analysis of the actin dynamics in this system. Moreover, the activation status of villin and its mutant activities in cells cannot be controlled. This prompted us to use a simplified biomimetic system.
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) and beads (van der Gucht et al., 2005) in the motility medium: an actin nucleator (Arp2/3) and its activator grafted around the beads (VCA), a capper (gelsolin or CapZ) and the depolymerizing/severing protein ADF/Cofilin. We replaced the capping protein of this assay by villin WT and RRI in the presence of 150 µM free calcium. In these conditions, WT villin is able to cap and sever actin filaments, whereas RRI villin is mostly able to cap filaments as shown in the pyrene assays. The presence of villin WT and RRI instead of the usual cappers allowed the formation of comets and the movement of the beads in the motility medium (Figure 7A). By phase contrast, the comets formed in the presence of the severing mutant of villin appeared darker and thus are denser than those formed in the presence of WT villin (refer also to Figure 7C). We analyzed the velocities of the beads induced by villin and its severing mutant. We measured the beads velocity by recording the lengthening of a comet over a given time. Indeed, as previously reported with this assay (Bernheim-Groswasser et al., 2002), the actin comets are terminated with a dense cloud at the end opposite to the bead. This dense part corresponds to the symmetry breaking event that precedes comet formation (van der Gucht et al., 2005) and is visible throughout the experiment. We thus took advantage of this effect to measure the velocity of the beads that coincides with the lengthening of the comet over time. In the presence of RRI villin, the velocity of the beads was reduced to <50% of the velocity induced by WT villin (Figure 7B). We decreased the free calcium concentration of the motility medium to 3 µM, conditions known to be saturating for the capping activity of villin whereas its severing activity is no longer detectable (Northrop et al., 1986). At 3 µM calcium, the velocity of the beads was not significantly higher for WT villin compared with RRI villin (Figure 7B). The average velocity of the beads remained about 0.2 µm/min in the presence of villin RRI but dropped from 0.45 to 0.2 µm/min in the presence of WT villin. The severing activity of villin is thus responsible for an enhancement of actin-based motility in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). There are several explanations for this discrepancy: villin and gelsolin are structurally similar; however, their specific activities are not regulated via identical mechanisms. Therefore the same mutations in the two proteins may have two different effects. In addition the gelsolin mutations were made on a truncated protein that may not mimic the behavior of a full-length protein with the same mutations. This highlights the importance of the various domains in the regulation of the proteins different activities. The severing mutant characterized in our work has nucleation and capping and bundling activities similar to that of WT villin and localizes properly in cells. The use of this mutant in functional assays in vivo and in vitro allows us to demonstrate that villin-severing activity is responsible for an enhancement of actin-based motility.
Villin-severing Activity Enhances Actin-based Motility
Villin increases the velocity of the bacteria S. flexneri in vivo (Athman et al., 2005). We not only confirm this result in MDCK cells by reporting a 45% rise in the average velocity of the bacteria in presence of WT villin but also show that the severing mutant of villin generated in this study abolishes this increase. The fact that the disassembly rate of the comets was not affected, whereas length and velocities were modified, is in favor of a role for villin in an enhancement of the polymerization at the surface of the bacteria. Similar results were obtained for L. monocytogenes motility after overexpression of gelsolin in infected cells (Laine et al., 1998). Nevertheless, a decrease, due to gelsolin, of the rate of Listeria actin tails disassembly has recently been observed after reduction of the calcium concentration (Larson et al., 2005). This was not accompanied by an impairment of the velocity of the bacteria. Playing on the regulation of gelsolin by calcium seems thus to have different effects than the ones observed previously by Laine et al. (1998) and in this work for villin.
Numerous parameters that cannot be evaluated are influencing actin polymerization and the movement of the bacteria in the cell. Among them, phosphorylation and phosphoinositides are known to modulate the activity of villin and likely of the RRI mutant. Moreover, the heterogeneity of the cytoplasm and the multiple obstacles that the bacteria have to face are probably responsible for the wide range of velocities reported (Figure 6B and Goldberg and Theriot, 1995) and for the variability observed in the length and intensity of the comet tails. We thus decided to mimic this movement in a simplified model system in order to control these parameters, especially the calcium-dependent biochemical activities of villin, and to validate that the effect reported in vivo is directly due to the lack of severing activity in RRI villin. The in vitro motility assay that we used generates the actin-based movement of beads. Despite slight differences with the in vivo situation (lower velocities, no detectable disassembly of the comets rear), the in vitro motility assay strongly strengthens the cell analysis as it results in a twofold increase in the speed of the beads in the presence of WT villin without influencing the disassembly of the comets. This confirms that the severing activity of villin by itself accounts for the enhanced motility of the bacteria. Apart from the fact that we cannot rule out contributions of other, uncharacterized activities of villin that may be affected by the mutations, our results strongly support the relevance of villin-severing activity in vivo.
What Mechanism Can Be Proposed from the In Vitro–reconstituted Motility?
This is the first report where villin is used to reconstitute an actin-based movement in vitro. We show that villin is able to replace the capping protein commonly used in this motility assay. The comparison of villin and its severing mutant RRI demonstrates a cooperation between the capping and severing activities in inducing and accelerating the actin-based movement. The comets formed in cell-free assays are most probably made of an Arp2/3 branched actin array (Svitkina and Borisy, 1999; Cameron et al., 2001). The site of polymerization is located at the interface between the bead and the comet where the Arp2/3 complex is activated. A capping protein, villin in our case, is required to limit the elongation process in space and to concentrate the force production toward the bead. Villin has a higher affinity for barbed end than for side binding on actin filaments (Northrop et al., 1986). Hence, as soon as the filaments elongate away from the bead, they are capped by villin, and all filaments that do not face the bead surface are expected to be barbed-end capped.
To account for the increased velocity reported, the severing activity of villin must somehow raise the concentration of actin monomers available for polymerization and hence accelerate the polymerization rate, which is directly proportional to the concentration of actin subunits (for review see Pollard and Borisy, 2003). As gelsolin, villin severs actin filaments and rapidly caps the newly formed barbed ends (Northrop et al., 1986). Severing by villin thus creates a net increase of free pointed ends. Consequently, in our assay, the villin-severing activity induces the appearance in the medium of an increased number of filaments capped at their barbed ends and free at their pointed ends. This increases net depolymerization and leads to a higher concentration of actin monomers. Actin polymerization is enhanced at the only free barbed-ends available, at the interface between the beads and the comets (illustrated in Figure 8A). Both in vitro and in vivo, there is no difference in the disassembly rate of the comets, whereas the increase in the velocities proves that actin cycling is enhanced by villin severing. Villin severing would thus not occur in the dense array constituting the comets but in a subset of other actin filaments. The different mechanisms controlling the disassembly of the structurally distinct actin arrays present in cells are indeed not well understood and are starting to be addressed (Brieher et al., 2006).
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Among the proteins used in this in vitro motility assay is the protein ADF/Cofilin. ADF/Cofilin used at high concentrations (5 µM) strongly accelerates the symmetry breakage of the actin gel growing around the beads, hence the appearance of a comet, and highly increases the velocity of bacteria or beads (Loisel et al., 1999; van der Gucht et al., 2005). Its property on actin filaments—depolymerizing and/or severing—is still controversial (Carlier et al., 1997; Zebda et al., 2000; Ichetovkin et al., 2002). Even if villin is able to sever filaments, replacing ADF by villin failed in producing motility: a short comet could only very rarely be observed with 100 nM villin in the absence of ADF but never at higher villin concentrations. The concentrations of villin allowing for the formation of comets in the presence of ADF are restricted to a very narrow range, as was already observed for CapZ (Loisel et al., 1999). The velocity of the beads picks at 50–100 nM villin and the movement is already blocked at 300 nM (data not shown). Consequently, at the concentrations used for ADF, the capping activity of villin overcomes the nucleation process by Arp2/3 and blocks the system as it has already been described for CapZ and gelsolin (Loisel et al., 1999; Pantaloni et al., 2000; van der Gucht et al., 2005).
Villin-severing Activity in Cell Movement
What clues does this work provide to the understanding of villin cellular function ? We demonstrated previously that villin enhances the motility and morphogenesis of cells (Athman et al., 2003). The increase in free barbed ends and the reduced F-actin content observed are in favor of a severing mechanism. As for villin, cell motility and dynamics have been positively correlated to cellular gelsolin content. Cells deficient in gelsolin exhibit defects in migration (Witke et al., 1995; Chen et al., 1996) and in cytoskeletal remodeling (Hartwig, 1992; Witke et al., 1995; Lu et al., 1997). In accordance with our observations for villin, increased velocity in fibroblasts expressing gelsolin correlates with reduced F-actin fraction and increased actin turnover (McGrath et al., 2000). Finally, villin as well as gelsolin were reported to accelerate the motility of S. flexneri (this work and Athman et al., 2005) and L. monocytogenes (Laine et al., 1998), respectively.
This set of data suggests a regulation of the actin cycle by the F-actin–severing activity of proteins like villin. However, because these proteins have multiple properties toward actin, at least in vitro, it remained to prove that the observed phenotypes are effectively due to the severing activity. This work is to our knowledge the first demonstration of a direct role of the severing activity of villin or a related protein in the enhancement of actin dynamics sustaining motility in vitro and in vivo.
We believe that this role of the severing activity of villin has a relevance in vivo for cell shape changes and movement. By creating pointed ends in an environment where filaments barbed ends are all capped, as in a lamella away from the extreme leading edge, villin could accelerate depolymerization and enhance actin polymerization rate at the leading edge. In vivo, on the contrary to our in vitro motility assay, villin can be dissociated from the barbed ends by several mechanisms as binding to phosphoinositides (Janmey and Matsudaira, 1988; Kumar et al., 2004b). Villin severing can then also contribute, in cooperation with other severing proteins such as gelsolin and ADF/Cofilin (Chan et al., 2000; Zebda et al., 2000), to the rapid creation of free barbed ends when a burst of polymerization is necessary as for the extension of a lamellipodium.
Finally, the restriction of villin expression to epithelial cells that develop a brush border hints at a particular role of villin in these specialized cells (Figure 8B). Intestinal epithelial cells move in response to signals or stresses in various situations, during embryogenesis, wound healing, or metastatic process. They are not adapted to move in their apico-basal polarized state but need to undergo an EMT to acquire a motile "fibroblastic-like" morphology (Nusrat et al., 1992). This transition requires the remodeling of the actin cytoskeleton. Gelsolin is absent from the microvilli (Yin et al., 1981), where villin is associated with the actin bundles. In response to signaling (calcium, phosphorylation; for review see Revenu et al., 2006), villin could efficiently shift from bundling to severing and rapidly break down this apically concentrated F-actin structure. It would contribute to the depolarization of the cell, increase the concentration of monomers available for polymerization of a new actin structure, and as reported here for bead or bacteria velocity, enhance the efficiency of movement. Our work thus supports a functional role of severing by villin in the EMT of enterocytes.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Sylvie Robine (sylvie.robine{at}curie.fr)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and RRI (
) villin plotted as a function of the concentration.
) and 1.8 nM for RRI villin (
). Inset, kinetics of actin filament barbed-ends elongation in the presence of various concentrations of RRI villin. (B) The nucleating activity was analyzed by evaluating the initial rate of polymerization of G-actin. Before inducing polymerization, pyrene–actin (2 µM) was incubated with villin WT and its variants (24 nM) in the presence of 20 µM calcium. No villin, polymerization of G-actin in the absence of nucleating protein; V1-3+HP, polymerization of G-actin in the presence of the nucleation mutant of villin V1-3+HP.
