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Originally published In Press as doi:10.1074/jbc.M605452200 on August 9, 2006

J. Biol. Chem., Vol. 281, Issue 41, 30393-30399, October 13, 2006
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A Direct Interaction between Actin and Vimentin Filaments Mediated by the Tail Domain of Vimentin*Formula

Osigwe Esue{ddagger}, Ashley A. Carson{ddagger}, Yiider Tseng§, and Denis Wirtz{ddagger}1

From the {ddagger}Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, the §Department of Chemical Engineering, University of Florida, Gainesville, Florida 32611, and the Howard Hughes Medical Institute graduate training program and Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, Maryland 21218

Received for publication, June 7, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microfilaments (F-actin),2 microtubules, and intermediate filaments (IFs) form extended networks that constitute the cytoskeleton and work in concert to drive dynamic cellular processes, including locomotion and division, and contribute greatly to the mechanical integrity of the cell (1, 2). The spatial organizations of these networks share a mutual interdependence: the disassembly of one network by pharmacological treatments often affects the organization of another (3–8). When IF assembly or organization is disrupted, the F-actin network is re-organized, whereas microtubule organization remains largely unaffected (9). On the other hand, the IF network collapses following microtubule disassembly and re-organizes upon actin depolymerization (3, 10).

Growing evidence suggests that cells have evolved specialized multibinding proteins that mediate the cross-linking of two or all three cytoskeleton filament networks. These proteins include fimbrin (11), the motor proteins kinesin (12), dynactin and dynein (13, 14), as well as members of the plakin family of large coiled-coil proteins (15, 16), including plectin (17) and BPAG1 (18, 19). Despite indirect evidence, it remains unclear whether major cytoskeleton filamentous proteins can interact directly, i.e. without auxiliary proteins.

IFs play a central role in helping cells resist mechanical stresses, maintaining the mechanical integrity of cells (20–22). Vimentin (57 KDa) is a type III IF found predominantly in cells of mesenchymal origin and a number of epithelial cell lines (23–26). Vimentin plays key roles in cell migration (27), proliferation (28), contractility (29, 30), as well as the gene expression profile of the cell when cells are subjected to fluid shear stress (31). Immunofluorescence microscopy indicates that vimentin structures colocalize with microfilaments and actin-containing structures (10, 32, 33). It has been speculated that this apparent interaction may be mediated through the tail domain of vimentin (10), but no evidence supports a direct interaction between actin and vimentin filaments. This may be in part due to the fact that traditional biochemical assays as well as light and electron microscopy are ill-suited to functionally test filament-filament interactions.

The tail domain of vimentin alone cannot form filaments (10, 34), but it interacts with its rod domain (35). The role of the IF tail domain in organizing vimentin and potentially F-actin networks is somewhat unclear. In vitro, the assembly and network formation of tailless ({Delta}T) vimentin filaments only appear compromised when there are mutations in the rod domain (9), whereas the ability of transfected vimentin to enter the nucleus is affected by its tail (36, 37). Transfected tailless Xenopus vimentin (36, 37) and human tailless keratin (38) have been shown to migrate into the nucleus of some cell lines, although this is not the case for human tailless vimentin, which does not migrate into the nucleus under similar conditions (36). These different cellular processes seem to rely on either direct or indirect interactions between vimentin and actin filament networks (10, 33).

Here, using purified proteins, we test the hypothesis that vimentin and actin filaments interact directly with each other to produce structures that are stiffer than the two individual networks they are made of. Like actin, vimentin can be assembled into filamentous structures upon addition of a polymerizing salt (39). Exploiting this property, actin and vimentin were polymerized simultaneously in the same buffer to form a mixed filamentous network.

Using quantitative rheometry, we measured the elasticity and viscosity of the resulting mixed networks of actin and vimentin filaments. The mechanical response of vimentin filament networks in the presence and absence of F-actin was assessed as a function of the protein composition of the mixed network, as well as the frequency and the amplitude of applied shear deformations (40). Our results show that a network containing both vimentin and actin filaments at a molar ratio of 3 to 1 displays an elasticity that is significantly higher and less frequency-dependent than the elasticity of networks containing only F-actin or vimentin filaments. {Delta}T vimentin, which assembles into filamentous structures of morphology similar to that formed by full-length (FL) vimentin, completely abrogates this synergistic effect. This suggests that the tail domain mediates a direct inter-filament interaction between vimentin IF and F-actin. Together with the fact that cells featuring prominent vimentin and F-actin networks are much stiffer than their counterparts lacking an organized F-actin network or vimentin network (41, 42), our results in vitro suggest that the mechanical synergy between actin and vimentin filaments is mediated by the tail domain of vimentin and may contribute to the overall mechanical response of cells and cytoskeletal cross-talk.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Purification—The purifications of human vimentin, {Delta}T vimentin, and the tail domain of vimentin are based on published protocols (10, 37, 39). Human vimentin, {Delta}T vimentin, and vimentin tail cDNAs were generous gifts from R. D. Goldman (Northwestern University Medical School), H. Herrmann (Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg), and M. W. Klymkowsky (University of Colorado, Boulder), respectively. Vimentin was dialyzed in a stepwise manner out of urea-containing buffer into 5 mM Tris-HCl (pH 8.4), 1 mM EDTA, 0.1 mM dithiothreitol. Actin was prepared from chicken breast (43) with an extra step of gel filtration by Sephacryl S-300 (Sigma) (44). Purified actin was stored as Ca2+-actin in continuous dialysis at 4 °C against buffer G (0.2 mM ATP, 0.5 mM dithiothreitol, 0.2 mM CaCl2, 1 mM sodium azide, and 2 mM Tris-HCl, pH 8.0).

In Vitro Filament Assembly—For assembly, both actin and vimentin were dialyzed into storage buffer (5 mM Tris-HCl, pH 8.4, 5 mM beta-mercaptoethanol, 0.2 mM ATP, 0.2 mM CaCl2, 1 mM sodium azide). Filaments were generated by adding 0.1 volume of 10x TKMEI (0.2 M Tris-HCl, 0.5 M KCl, 10 mM MgCl2, 10 mM EGTA, 100 mM imidazole, pH 7.0) polymerizing salt to 0.9 volume of unpolymerized protein in storage buffer.

Rheology—The mechanical properties of networks of actin and vimentin filaments were measured using a strain-controlled, 50-mm diameter cone-and-plate rheometer (ARES-100, TA Instruments, New Castle, DE) (45, 46). Shear deformations of controlled amplitude and frequency were applied by precise dynamic rotations of the lower plate of the rheometer. The upper cone of the rheometer is coupled to a computer-controlled motor, which applies either steady or oscillatory shear deformations of controlled frequency and amplitude. The cone is connected to a torque transducer, which measures the stress induced in the protein solutions by the applied shear deformations. The gelation of the filament networks was monitored by measuring the time-dependent elasticity. Elasticity was measured by applying two oscillatory deformations of 1% amplitude at a frequency of 1 rad/s every 120 s until a steady state was attained (~1–3 h). At steady state, the frequency-dependent viscoelastic moduli of the filament networks, G'({omega}) and G''({omega}), were computed by dividing the in-phase and out-of-phase components of the measured oscillatory stress induced within the network by the applied strain amplitude. The phase angle, {delta} = tan–1(G''/G') was also calculated.

Next, oscillatory deformations of small amplitude (1%) and frequency between 0.01 and 100 rad/s were applied to measure simultaneously the frequency-dependent elastic modulus, G'({omega}), and frequency-dependent viscous modulus, G''({omega}), of the filament networks. Finally, deformations of fixed frequency (1 rad/s) and amplitude between 0.1 and 1000% were applied to measure the elastic and viscous moduli as a function of the amplitude of the input shear deformation, g0. The statistical significance between moduli of networks of different protein composition was verified by pairwise comparison using the Student's t test, which yielded values of p < 0.05.

Electron Microscopy—The ultrastructure of negatively stained vimentin and actin filaments was examined by electron microscopy. Polymerizing vimentin and actin solutions were incubated in assembly buffer (25 mM Tris buffer, pH 7.4) obtained by mixing 1 volume of polymerizing buffer to 9 volumes of storage buffer. 10 µl of protein solution was placed on each collodion-coated electron microscopic grid. Grids were washed with assembly buffer and stained with 2% uranyl acetate solution (47). Electron microscopy was performed at the Integrated Imaging Center in Johns Hopkins University with a Philips 410 transmission electron microscope at magnifications between 65,000x and 105,000x, as indicated in the figure captions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Actin and Vimentin Filaments Interact Directly—We hypothesized that vimentin and actin filaments interact directly, i.e. without auxiliary proteins, forming structures that are stiffer than the two networks they are made of. Exploiting the unique assembly properties of vimentin among IFs, we co-polymerized vimentin and actin filaments in the same buffer, and monitored the viscoelastic properties of the mixed networks. Using a cone-and-plate rheometer, the elastic modulus, G' (which measures the propensity of the polymers to rebound after shear deformation), and the viscous modulus, G'' (which measures how much the specimen can flow under stress), of the protein solutions were measured upon onset of filament assembly.

The relative concentration of vimentin and actin was varied for a total protein concentration of 24 µM. The steady-state elastic modulus for the mixed filament networks reached a distinct maximum in stiffness of G'~ 40 dyn/cm2 for [actin] = 6 µM and [vimentin] = 18 µM, compared with 6 dyn/cm2 fora24 µM F-actin network (no vimentin present) and 15 dyn/cm2 for a 24 µM vimentin filament network (no actin present) (Fig. 1, A and B). Therefore, a mixed filament network containing one-fourth actin and three-fourths vimentin was stiffer than the networks containing only F-actin or only vimentin filaments.


Figure 1
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  		FIGURE 1.
Gelation kinetics of vimentin/actin filament networks. A, time-dependent, and B, steady-state elastic modulus, G' of mixed vimentin/actin filament networks. The total protein concentration was 24 µM for all concentration ratios tested. A well defined maximum G' value occurs at [vimentin] = 18 µM and [actin] = 6 µM, indicating enhanced interactions between filamentous proteins. * in B indicates that the difference between measured network elasticity and the elasticity of a 24 µM vimentin filament network is significant, as verified by pairwise comparison using the Student's t test. Symbols in A and B correspond to 24 µM actin (closed squares), 24 µM vimentin (open circles), and 6 µM actin and 18 µM vimentin (closed circles). C, phase angle, {delta}, of vimentin/actin filament networks as a function of total protein concentration. A phase angle of 90° describes the rheological behavior of a liquid (e.g. glycerol); a phase angle of 0° describes an elastic solid (e.g. a stiff rubber). The phase angle for the various actin/vimentin concentration ratios tested increased with actin concentration indicative of a more liquid-like rheological behavior. Symbols in B and C correspond to input shear frequencies of 1 rad/s (open squares) and 40 rad/s (closed squares) (see text for details). D, EM image of equimolar vimentin/actin filament network. Total concentration, 2 µM. Scale bar, 0.2 µm. There are two peaks in the distribution of filament diameters, 7.7 ± 1.4 nm and 10.8 ± 2.2 nm (mean ± S.D.), corresponding to the nominal diameters of actin and vimentin filaments, respectively.

 
Vimentin filament networks in the absence of actin displayed a low phase angle, {delta} ~ 5° (Fig. 1C), which corresponds to a solid-like rheological behavior, close to that displayed by a stiff rubber. The phase angle describes the delay between the imposed (input) deformation and the measured (output) mechanical stress induced by that deformation in the network. This delay is minimum (0°) for an elastic solid-like rubber and maximum (90°) for a viscous liquid-like glycerol. As the actin-to-vimentin molar ratio increased, mixed vimentin/actin networks became significantly more liquid-like, reaching a phase angle of ~30° for an F-actin network containing no vimentin (Fig. 1C).


Figure 2
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  		FIGURE 2.
Dynamic properties of FL vimentin/actin filament networks at steady state. A, frequency-dependent elastic modulus, G'({omega}), of FL vimentin/F-actin networks as a function of protein concentration. Symbols correspond to 24 µM actin (closed squares), 24 µM vimentin (open squares), and 6 µM actin and 18 µM vimentin (closed diamonds). B, concentration-dependent exponent a, which was extracted from power law fits, G'~ {omega}a, of profiles such as shown in A. The exponent a increased as the molar ratio of actin to vimentin increased, which suggests that the filamentous networks became more dynamic, i.e. there was more relative sliding among filaments, for increasing actin concentration in mixed vimentin/actin filament networks.

 
Through electron micrographs of negatively stained suspensions of either pure F-actin or pure vimentin filament network, we verified that the present buffer conditions did not affect their normal morphology. EM was also used to examine the structures formed by the copolymerization of actin and vimentin (Fig. 1D). Analysis of micrographs of individual filaments in mixed vimentin/actin filament networks resulted in a distribution of filament diameters with two narrow peaks confirming the presence of separate actin and vimentin filaments within the network. The mean ± S.D. values of the distributions were 7.7 ± 1.4 nm and 10.8 ± 2.2 nm, which correspond to the known diameters of actin and vimentin filaments, respectively. Therefore, each filament in the mixed filament networks contained only one type of subunit.

Dynamic Response of Vimentin/Actin Filament Networks—For a fixed polymer length and polymer concentration, the mobility of filaments in a network depends mostly on their potential to interact through cross-linking interactions (48). To assess the extent of mobility of the filaments within entangled vimentin/actin filament networks, these networks were subjected to shear deformations of increasing frequency. The elastic modulus of F-actin networks (no vimentin present) increased steadily with increasing rates of shear, i.e. with increasing shear frequency (Fig. 2A). This result indicates that actin filaments had progressively less time to relax the mechanical stress imposed by the rheometer for increasing rates of shear rates. However, by reducing actin concentration and increasing vimentin concentration in mixed networks, we observed a significantly reduced frequency dependence of the elastic modulus (Fig. 2A). This indicates that filaments in mixed vimentin/actin filament networks became less mobile than in networks containing only F-actin. Therefore, these mixed filament networks were less capable of relaxing mechanical stresses, presumably due to enhanced interactions between actin and vimentin filaments (Fig. 2A).

These frequency-dependent elasticity profiles (Fig. 2A) could be fit approximately to power laws of the type G'({omega}) ~ {omega}a, with an exponent a that describes the steepness of the frequency dependence of G'({omega}). The exponent a increased for increasing actin content in mixed networks, which suggests that there was more relative movements among filaments in actin-rich networks than in vimentin-rich networks (Fig. 2B).

The Tail Domain of Vimentin Mediates Interactions between Vimentin and Actin Filaments—We hypothesized that the tail domain of vimentin mediates the synergistic enhancement of elasticity observed in mixed vimentin/actin filament networks. Therefore we repeated our rheological assays for mixed suspensions of actin and {Delta}T vimentin filaments. The steady-state elastic modulus of networks containing both actin and {Delta}T vimentin filaments decreased as the molar ratio of actin to vimentin in the mixture increased. However, unlike their FL vimentin/actin filament network counterparts, the relative elastic modulus (G'/G'vim = 24µM) of {Delta}T vimentin/actin filament networks displayed no distinct maximum for an intermediate protein composition (Fig. 3A). Moreover, the higher phase angle value of the networks suggested that actin and {Delta}T vimentin filaments formed more liquid-like networks compared with actin and FL vimentin filaments over the same concentration range (Fig. 3B).

We further tested whether the absence of mechanical synergy between {Delta}T vimentin and actin filaments correlated with diminished inter-filament interactions by analyzing the frequency-dependent elasticity, G'({omega}). Although the exponent a from power-law fits of G'({omega}) also increased with actin concentration, it was higher in the presence of {Delta}T vimentin than in the presence of FL vimentin (Fig. 3C). This suggests that {Delta}T vimentin allowed significantly more inter-filament sliding in mixed networks than their FL vimentin counterparts. EM studies did not show ultrastructural differences between FL vimentin/actin and {Delta}T vimentin/actin filament networks (Fig. 3D). Hence the observed difference in the mechanical response of mixed filament protein networks is probably due to direct interactions between vimentin and actin filaments (Fig. 3).

Binding studies showed an interaction between F-actin and the tail domain of vimentin (supplemental Fig. S1). We evaluated the binding kinetics of vimentin tails to F-actin using a pelletting assay (49). Solutions of actin monomers of fixed concentration were mixed with various concentrations of vimentin tails before initiating actin filament assembly. The pellet recovered from a high speed centrifugation of the polymerized proteins was analyzed on SDS-gel electrophoresis, and the concentration of free (unbound) tails in the supernatant was measured. Analysis of the Coomassie-stained protein gels showed the presence of actin and vimentin tails in the pellet. A fit of the binding curve using nonlinear regression (supplemental Fig. S1) yielded an equilibrium dissociation constant, kd, of ~13.8 µM, indicative of weak binding between F-actin and the tail domain of vimentin.

The Mechanical Properties of {Delta}T and FL Vimentin Filaments Are Similar—The tail domain of vimentin does not affect vimentin assembly in vitro and in vivo (34, 37), hence we hypothesized that it would have negligible effects on the mechanical properties of vimentin networks. We compared the mechanical properties of FL and {Delta}T vimentin filament networks. EM studies showed that both FL vimentin and {Delta}T vimentin formed filaments of similar morphology (Fig. 4) (34). There was a negligible difference in elastic moduli of both FL and {Delta}T vimentin filament networks (no actin present) over the tested concentration range (Fig. 4A). The elastic moduli of both networks increased weakly with protein concentration, C, scaling as G'(C) ~ C0.37, and G'(C) ~ C0.35 (estimated at a frequency of 1 rad/s), for FL and {Delta}T vimentin, respectively. Polymer theory suggests that this concentration dependence is consistent with cross-linked flexible filaments (50).


Figure 3
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  		FIGURE 3.
Mechanical properties of {Delta}T vimentin/actin filament networks. A, relative elastic modulus of F-actin networks mixed with either FL (closed squares) or {Delta}T (open squares) vimentin is plotted as a function of protein concentration. The absence of a maximum in the elasticity of {Delta}T vimentin/actin filament networks suggests that the tail domain of vimentin mediates a direct interaction between vimentin and actin filaments. Elastic moduli were normalized by their value for a 24 µM network containing only vimentin. B, phase angle, {delta}, of vimentin/actin filament networks versus protein concentration. Actin and {Delta}T vimentin filaments formed more liquid-like networks compared with networks containing actin and FL vimentin filaments over the same concentration range. Symbols correspond to FL vimentin/F-actin (closed squares) and {Delta}T vimentin/F-actin (open squares). C, exponent a, extracted from power-law fits of G'({omega}), is plotted as a function of protein concentration for both FL/actin and {Delta}T vimentin/actin filament networks. Although the exponent increased with actin concentration for both mixtures, it was higher in networks containing {Delta}T vimentin than in those containing FL vimentin. This suggests that {Delta}T vimentin/actin filament networks allowed relatively more filament sliding than in their FL vimentin/actin counterparts. D, EM image showing a network of actin copolymerized with either equimolar FL or {Delta}T vimentin. Total concentration, 2 µM. Scale bars, 0.2 µm.

 


Figure 4
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  		FIGURE 4.
Vimentin filament elasticity in the presence/absence of its tail domain. A, comparison of the concentration-dependent elastic modulus, G', and B, phase angle, {delta}, of vimentin filament networks (FL vimentin is indicated by black bars, {Delta}T vimentin by gray bars). No actin was present in the solutions. The elasticity scales as G'(C) ~ C 0.37, and G'(C) ~ C 0.35 for FL and {Delta}T vimentin filament networks, respectively. C, EM image of FL vimentin and {Delta}T vimentin (inset) filament networks. Concentration, 2 µM. Scale bar, 0.2 µm.

 
The value of the phase angle also suggested that both networks harbored a strong solid-like character, with FL vimentin being slightly stiffer that {Delta}T vimentin (Fig. 4B). As the networks gelled, we tested the mobility of FL and {Delta}T vimentin filaments in networks, by exposing the networks to shear deformations of increasing frequency {omega} before a steady-state elasticity was reached (Fig. 5, A and B). The frequency-dependent gelation profiles were fit to power laws, G'({omega}) ~ {omega}a, and an exponent a was extracted from the plots shown in Fig. 5 (A and B). The measured value of the exponent a indicated that inter-filament interactions were similar for both FL and {Delta}T vimentin networks. Although a was slightly lower for FL vimentin (0.12 ± 0.01) than for {Delta}T vimentin (0.16 ± 0.01) filament networks (mean ± S.D.), they had a parallel downwards trend, which indicated that inter-filament interactions grew stronger with time. Together these results suggest that networks of {Delta}T vimentin filaments have mechanical properties similar to those of networks of FL vimentin filaments.

By subjecting vimentin filament networks to increasing strain amplitudes, we found that the amplitude of the deformation at which vimentin networks began to yield and break down (point where the elastic modulus dropped by 10% of its original value) was independent of vimentin concentration (data not shown). Unlike keratin IF 5/14 network, whose mechanical resilience is significantly affected when keratin-14 tail domain is truncated (51), the truncation of the tail domain of vimentin did not affect the mechanical resilience of its network. Moreover, FL and {Delta}T vimentin filaments formed networks of similar ultrastructures, as assessed by EM and negative staining (Fig. 5C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The three major cytoskeleton proteins (F-actin, microtubule, and IF) form networks that are mutually affected by the re-organization or disassembly of any one of them. Immunofluorescence microscopy shows that FL vimentin and the vimentin tail localize near actin networks in cells, suggesting an interaction between vimentin and actin filaments mediated by the tail domain of vimentin (10). However, this interaction could be indirect, i.e. mediated by auxiliary proteins.

We report the strongest evidence yet of a direct interaction between two of the three major cytoskeletal networks, vimentin and actin, which has remarkable effects on their network mechanical features. Actin and vimentin are copolymerized simultaneously and the viscoelasticity of the resulting filament networks is assessed by quantitative rheology. Unlike mixed IF proteins (e.g. desmin and vimentin (52)), which can form filaments containing both subunits, mixed actin and vimentin networks contain filaments with only one type of subunit. The mixed vimentin/actin filament network displays an elasticity that does not uniformly interpolate between the elasticity of networks of pure F-actin and that of pure vimentin IF. Analysis of the rheology measurements shows that a maximum in network stiffness occurs at an actin:vimentin ratio of 1:3 and that the tail domain of vimentin mediates inter-filament interactions between F-actin and filamentous vimentin.

A binding assay showed that the vimentin tail binds F-actin with a Kd of ~14 µM. This equilibrium dissociation constant is relatively high, but similar to dissociation constants of the cross-linking protein {alpha}-actinin for F-actin (44). Like other F-actin cross-linking proteins, vimentin does not require a high affinity for F-actin. Indeed, the interactions between F-actin and vimentin tails are multiplied by the high number of entanglements formed between each vimentin filament and the actin filaments in a dense network. These entanglements, reinforced by direct interactions between vimentin tails and actin filaments, prevent easy sliding of vimentin filaments when the network is under mechanical stress. This inhibited filament movement induces a high network elasticity, higher than in networks of actin and tailless vimentin filaments, which do not interact directly with each other.

In the absence of mutations in the rod domain, {Delta}T vimentin assembles into filaments that appear identical to those formed by FL vimentin (9), as assessed by conventional electron microscopy. However, there are also reports that tailless vimentin filaments are slightly thicker than FL vimentin filaments (34). In vivo, the presence of the tail domain of vimentin appears to affect its ability of transfected Xenopus vimentin to localize inside the nucleus of cells (36, 37). This suggests that, within the cell, the tail domain could affect the organization of vimentin. In many cell types, IFs appear to align more preferably with microtubules than with actin structures, presumably due to stronger interactions between microtubules and IFs (10). However, isolated vimentin tails colocalize with actin networks and not with microtubule networks, which suggests that vimentin tails have a higher affinity for F-actin structures and that microtubule-IF interaction is not mediated by the IF tail domain.


Figure 5
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  		FIGURE 5.
The tail domain of vimentin mediates inter-filament interactions in vimentin/actin filament networks. A, FL and B, {Delta}T vimentin filament networks were subjected to shear deformations of 1% amplitude and increasing frequency at various times during gelation until steady-state elasticity was attained. Protein concentration was 18 µM (~1 mg/ml FL vimentin). Symbols correspond to 10 min (closed circles), 30 min (open circles), 60 min (closed squares), 120 min (open squares), 180 min (closed diamonds), and 240 min (open diamonds) after the onset of gelation. C, exponent a, which was extracted from power law fits of G'({omega}) and plotted as a function of gelation time. The final value of the exponent a, a = 0.12 ± 0.01 and 0.16 ± 0.01 (mean ± S.D.) for FL and {Delta}T vimentin filament networks, respectively, indicated that inter-filament interactions were similar.

 
Scanning transmission EM of unstained intermediate filaments (including keratin, vimentin, and desmin) reveals that IFs have filament widths of ~16 nm, which is larger than the 10 nm filament width measured using conventional heavy metal stain EM (53, 54). Cary et al. (10) propose that the excess 6 nm is possibly occupied by the head and tail domain, which projects out of the central axis of its filament. The hypothesized projection of nonhelical IF tails is similar to the observed projection of neurofilaments tails of their NF-H and NF-M subunits (55). Antibodies against NF-H and NF-M subunits localize to the peripheral as well as cross-bridges of NFs (56–58). Bousquet et al. (51) also showed that the nonhelical tail domain of keratin K14 facilitates lateral filament association with keratin filaments forming bundles in vivo and gives the structures high mechanical attributes. These putative outward projecting vimentin tails may display the proper orientation to promote strong interactions with F-actin. Another mechanism of synergistic interaction between actin and vimentin filaments could be that the tails mediate electrostatic interactions that would influence the overall mechanical properties of mixed actin and vimentin filament networks. These observations together with our results suggest a direct interaction between F-actin and vimentin filaments mediated by the tail domain of vimentin, which results in an enhanced elasticity of mixed vimentin/actin filament in networks.

For a constant protein concentration, polymer theory predicts that the elasticity of a polymer network depends weakly on the intrinsic rigidity of the filaments (i.e. their persistence length) and depends mostly on inter-filament interactions between filaments. Increasing the rate of shear on a network interrogates the ability of filaments within the network to slide past one another. Actin filaments slide past each other much more readily than vimentin filaments. In further support of strong inter-filament interactions, vimentin structures also exhibit a low phase angle (e.g. the phase angle of water is 90°, whereas that of rubber is 0°) compared with F-actin. On the other hand, actin forms filament networks with a higher phase angle that decreases as the ratio of vimentin within the network increases.

Vimentin assemble into filaments with or without their tail domain, which suggests that vimentin assembly is not mediated by its tail. The elastic modulus of FL vimentin filament networks is not significantly higher in the absence of its tail, which implies that vimentin network elasticity does not stem from the interaction of its tail. This indicates that our observed enhanced stiffness of FL vimentin/actin filament networks does not simply stem from enhanced content in FL vimentin filaments in mixed networks.

Our study does not identify the vimentin-binding domain of F-actin, but our data strongly suggest that there is a direct interaction between the tail domain of vimentin and F-actin, which mediates a significant enhancement of the mechanical properties of mixed actin/vimentin filament networks compared with networks containing only F-actin or only vimentin.


   FOOTNOTES
 
* This work was funded by NASA Grant NAG9-1563, National Institutes of Health Grant GM075305, and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Dept. of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-7006; Fax: 410-516-5510; E-mail: wirtz{at}jhu.edu.

2 The abbreviations used are: F-actin, filamentous actin; IF, intermediate filament; FL, full-length; {Delta}T, tailless; EM, electron microscopy. Back


   ACKNOWLEDGMENTS
 
We thank Dr. R. D. Goldman (Northwestern University Medical School), Dr M. W. Klymkowsky (University of Colorado, Boulder), and Dr. H. Herrmann (Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg) for generous gifts of cDNAs of human vimentin, the tail domain of vimentin, and human {Delta}T vimentin, respectively. We also thank Michael McCaffery of the Integrated Imaging Center at Johns Hopkins for technical assistance.



   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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