Intriguing Self-Assembly of Large Granules of F ... - ACS Publications

We report microscopic observations and a structural determination of actin granules self-assembled in concentrated solutions of actin filaments (F-act...
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Langmuir 2005, 21, 2789-2795

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Intriguing Self-Assembly of Large Granules of F-Actin Facilitated by Gelsolin and r-Actinin Jay X. Tang,*,† Hyeran Kang,† and Jinfeng Jia‡ Physics Department, Brown University, 184 Hope Street, Providence, Rhode Island 02912, and State Key Laboratory for Surface Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China Received November 13, 2004. In Final Form: January 29, 2005 We report microscopic observations and a structural determination of actin granules self-assembled in concentrated solutions of actin filaments (F-actin). Optical microscopy shows reproducible formation of numerous and stable granules of densely packed F-actin of variable sizes on the order of 10 µm. These granules coexist with a uniform network of F-actin of a lower concentration. The microscopic segregation of F-actin into two distinct states is assisted by an actin cross-linking protein, R-actinin. The rapid on and off rates and temperature sensitivity of the R-actinin/F-actin interaction facilitate the formation of multimicrometer-sized granules of well-defined shapes. Additional physical factors such as the excluded volume effect and the minimization of surface energy act in concert with the specific molecular interactions to define the intriguing granular formation. Both the biochemical specificity of R-actinin and the thermodynamics of phase transitions are required for understanding such large scale self-assembly.

Introduction Protein complexes formed by the cytoskeletal filaments F-actin and a number of actin binding proteins play essential roles in cellular and tissue biology, such as muscle contraction, cellular shape change, and migration.1 To complement extensive biochemical studies of actin and actin binding proteins at low concentrations, we investigate the self-assembly properties of F-actin at concentrations of 20 mg/mL and higher, comparable to its presence in the leading edge of motile animal cells and in muscle fibers. Actin purified from the muscle tissue exists in low ionic strength solutions as globular monomers, termed G-actin. Addition of salt induces the polymerization of G-actin into long (up to tens of micrometers), thin (diameter 8-10 nm),2 and stiff (persistence length over 15 µm)3,4 filaments. The polymerized filaments of actin can form an entangled isotropic network,5 a nematic liquid crystalline state,6-8 and paracrystalline bundles mediated by either polyvalent cationic agents9 or specific actin crosslinking proteins.10 * Corresponding author. Tel: 401 863 2292. Fax: 401 863 2024. E-mail: [email protected]. † Brown University. ‡ Chinese Academy of Sciences. (1) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. Molecular biology of the cell, 4th ed.; Garland, New York, 2002. (2) Holmes, K.; Popp, D.; Gebhard, W.; Kabsch, W. Atomic model of the actin filament. Nature 1990, 347, 44-49. (3) Gittes, F.; Mickey, B.; Nettleton, J.; Howard, J. Flexural Rigidity of Microtubules and Actin Filaments Measured from Thermal Fluctuations in Shape. J. Cell Biol. 1993, 120, 923-934. (4) Isambert, H.; Venier, P.; Maggs, A. C.; Fattoum, A.; Kassab, R.; Pantaloni, D.; Carlier, M. Flexibility of Actin Filaments Derived from Thermal Fluctuations. J. Biol. Chem. 1995, 270, 11437-11444. (5) Janmey, P. A.; Hvidt, S.; Kas, J.; Lerche, D.; Maggs, A.; Sackmann, E.; Schliwa, M.; Stossel, T. P. The mechanical properties of actin gels. Elastic modulus and filament motions. J. Biol. Chem. 1994, 269, 3250332513. (6) Furukawa, R.; Kundra, R.; Fechheimer, M. Formation of liquid crystals from actin filaments. Biochemistry 1993, 32, 12346-12352. (7) Suzuki, A.; Maeda, T.; Ito, T. Formation of liquid crystalline phase of actin filament solutions and its dependence on filament length as studied by optical birefringence. Biophys. J. 1991, 59, 25-30. (8) Viamontes, J.; Tang, J. X. Continuous isotropic-nematic liquid crystalline transition of F-actin solutions. Phys. Rev. E: Stat., Nonlin., Soft Matter Phys. 2003, 67, 040701-040704.

R-Actinin is a common actin cross-linking protein in both muscle and nonmuscle cells.11 In the sarcomere, R-actinin is concentrated near the z-line, which actually defines a cross-sectional plane to which the thin filaments are attached. In nonmuscle cells, such as NIH 3T3 fibroblasts, R-actinin is highly concentrated in stress fibers, as well as focal adhesion sites. In all these cellular settings the cross-linking activity of R-actinin is essential for various functions. R-Actinin consists of an actin binding domain as the head and four domains in linear repeat as its tail.11,12 Its biochemically functional state has been shown as an antiparallel dimer, with an actin binding site on each end and the tail repeats essentially overlapping in an antiparallel fashion. As a result, the R-actinin dimers are capable of cross-linking two actin filaments of variable crossing angles. In vitro, a substoichiometric amount of R-actinin has been shown to enhance the elasticity of an F-actin network,13-15 although the gelation effect of R-actinin is weaker than that of filamin.16 The binding of R-actinin/F-actin is highly dynamic, with fast on and off rates.13-15,17 This unique property is likely relevant to its (9) Tang, J. X.; Janmey, P. A. Polyelectrolyte Nature of F-actin and Mechanism of Actin Bundle Formation. J. Biol. Chem. 1996, 271, 85568563. (10) Otto, J. J. Actin-bundling proteins. Curr. Opin. Cell Biol. 1994, 6, 105-109. (11) Blanchard, A.; Ohanian, V.; Critchley, D. The structure and function of alpha-actinin. J. Muscle Res. Cell Motil. 1989, 10, 280-289. (12) Djinovic-Carugo, K.; Young, P.; Gautel, M.; Saraste, M. Structure of the alpha-actinin rod: molecular basis for cross-linking of actin filaments. Cell 1999, 98, 537-546. (13) Wachsstock, D. H.; Schwartz, W. H.; Pollard, T. D. Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels. Biophys. J. 1993, 65, 205-214. (14) Wachsstock, D. H.; Schwarz, W. H.; Pollard, T. D. Cross-linker dynamics determine the mechanical properties of actin gels. Biophys. J. 1994, 66, 801-809. (15) Xu, J.; Wirtz, D.; Pollard, T. D. Dynamic cross-linking by alphaactinin determines the mechanical properties of actin filament networks. J. Biol. Chem. 1998, 273, 9570-9576. (16) Bennett, J. P.; Zaner, K. S.; Stossel, T. P. Isolation and some properties of macrophage R-actinin: evidence that it is not an actin gelling protein. Biochemistry 1984, 23, 5081-5086. (17) Kuhlman, P. A.; Ellis, J.; Critchley, D. R.; Bagshaw, C. R. The kinetics of the interaction between the actin-binding domain of alphaactinin and F-actin. FEBS Lett. 1994, 339, 297-301.

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in vivo functions. For instance, addition of a few micromolar R-actinin into a motility medium with an essential set of proteins (actin, Arp2/3 complex, cofilin, and a capping protein) increases the growth rate of actin tails stimulated by the WASP coated beads.18 The binding of R-actinin/ F-actin is also sensitive to temperature, with the affinity stronger by severalfold as the temperature is lowered from room temperature to 4 °C.15,17,19 Although the physiological significance of this temperature sensitivity is unclear, previous in vitro studies have used this property to manipulate the rheological behavior of the R-actinin/ F-actin network.16 In this study, we report assembly of giant actin/R-actinin granules, most of which have the well-defined shape of American footballs and a molar ratio between 1:16 and 1:20 for R-actinin/F-actin inside the granules. We found that these granules only form in a solution of high actin concentration (20 mg/mL or higher), with the R-actinin content no more than a few percent of actin by weight. The dynamic formation and growth of these actin granules are facilitated by a moderate increase of temperature, such as incubation at 37 °C for several hours, suggesting that a weaker or more transient binding is more effective in facilitating the granular formation. Regulating the length of actin filaments with gelsolin,20 a filamentsevering and end-capping protein, further promotes the growth of numerous granules. All these properties together suggest a mechanism of dynamic self-assembly to form novel structures, a mechanism richly exploited in the complex multicomponent cellular environment. Materials and Methods Sample Preparation. Monomeric (G-)actin was purified from the rabbit back muscle according to the method of Pardee and Spudich,21 using Sephadex G-150 resins in the final size exclusion chromatography step (Pharmacia Biosciences). The nonpolymerizing buffer contained 2 mM Hepes, adjusted to pH 7.5, 0.2 mM CaCl2, 0.5 mM DTT, 0.5 mM ATP, and 0.5 mM NaN3. F-Actin was polymerized from G-actin by addition of 2 mM MgCl2 and 50 mM KCl, unless specified otherwise. All chemicals were purchased from Sigma (St. Louis, MO). Skeletal muscle R-actinin was purchased from Cytoskeleton (Denver, CO). Recombinant human gelsolin was a kind gift from Paul Janmey, University of Pennsylvania Medical School. To grow actin granules, substoichiometic amounts of gelsolin (1:185-500 of actin) and R-actinin (1:100-400 of actin) were added into G-actin prior to its polymerization. Concentrated samples containing predominantly tactoidal granules were prepared by two different methods, both starting from an initial F-actin concentration of typically 5-10 mg/mL. In one method, actin filaments were sedimented by ultracentrifugation at over 100000g for 1 h at a temperature between 10 and 25 °C, followed by resuspension of the pellet to a calculated final actin concentration in the range of 20-100 mg/mL. The resuspension was then incubated at 37 °C for several hours to allow formation of the granules. The number of the granules increased with the incubation time, as did the average size of the granules, albeit slowly. In the second method, the solution was spun at a relatively low speed (10000g) for several hours. After two-thirds of the dilute sample on the top was removed, the compressed gel was allowed to reequilibrate at 1g overnight. The success of the second method in generating actin granules was aided by the fact that (18) Loisel, T. P.; Boujemaa, R.; Pantaloni, D.; Carlier, M. F. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 1999, 401, 613-616. (19) Goll, D. E.; Suzuki, A.; Temple, J.; Holmes, G. R. Studies on purified alpha-actinin I. Effect of temperature and tropomyosin on the alpha-actinin/F-actin interaction. J. Mol. Biol. 1972, 67, 469-488. (20) Janmey, P. A.; Hvidt, S.; Peetermans, J.; Lamb, J.; Ferry, J. D.; Stossel, T. P. Viscoelasticity of F-actin and F-actin/gelsolin complexes. Biochemistry 1988, 27, 8218-8227. (21) Pardee, J. D.; Spudich, J. A. Purification of muscle actin. Methods Enzymol. 1982, 85, 164-181.

Tang et al. the sample was warmed considerably during hours of tabletop spinning in the ambient environment. This was not recognized early in the study but was confirmed later when the same procedure was repeated with the tabletop centrifuge placed in a ventilated 4 °C environment. Repeated preparations through which samples were kept at 4 °C produced thin F-actin/R-actinin bundles but few granules. Note also that some earlier observations were made using actin prepared without the column purification step, which contained small and variable amounts of actin binding proteins such as R-actinin and perhaps also capping proteins. Therefore, we observed formation of actin granules even though neither gelsolin nor R-actinin was added in an earlier study. Granular formation was further exploited by a number of variations in the preparation. Of particular note is that when repeated sedimentation and resuspension cycles were applied, we observed more complicated structures such as small granules embedded within large granules or tubules. All structures formed appeared rather stable upon dilution by the same F-actin buffer solution. Electron Microscopy. Small actin granules were subject to electron microscopy (EM) following a negative staining procedure. The granules were stained with 1% uranyl acetate, rinsed, and dried in the hood before EM imaging. Images were acquired on a Phillips CM200 (120 kV) using a Gatan MSC CCD camera (1K × 1K pixels, with each pixel reading stored in 12 bits), available through the Imaging Technology Group of Beckman Institute, University of Illinois Urbana-Champaign. Optical Microscopy. Phase contrast optical micrographs were taken using a Nikon TE-300 with either a 40× objective (Figure 1) or a 60× oil objective (Figures 4-6). The fluorescence images (Figure 2) were taken using a Delta Vision deconvolution microscope. We found that the deconvolution analysis introduced artificial results to the images of individual slices for this particular application, and therefore only the unprocessed images of single slices are shown. Three different dye conjugates were used for triple staining of the actin tactoids. Coumarin phenyl isothiocyanate (CPITC)phalloidin was purchased from Sigma. Fluorescein chemically coupled to gelsolin (FITC-gelsolin) was a kind gift from Dr. Phil Allen of Hematology Division, Brigham and Women’s Hospital, Harvard Medical School. Tetramethylrhodamine chemically coupled to R-actinin (TRITC-R-actinin) was purchased from Cytoskeleton. A fluorescence quantification method was used to determine the actin and R-actinin concentrations inside the tactoids. Briefly, F-actin was partially (0.01% in molar ratio) labeled by adding rhodamin (TRITC) tagged phalloidin either prior to formation of the tactoidal granules or to a solution containing the tactoids. In either case, the sample was incubated overnight following the addition of the TRITC-phalloidin. The fluorescence intensity of large tactoids was measured and calibrated against a separate F-actin sample of 5 mg/mL, labeled with a factor of 20 larger in the TRITC-phalloidin to actin molar ratio (0.2%, as opposed to 0.01% for imaging the dense tactoids). Both the tactoids and the separate F-actin samples were thicker than the depth of focus and the thickness of the path over which the fluorescence signal was integrated by the 60× oil objective lens. The calibration was also designed so that the measured intensities of the two samples were comparable (after some initial trial-and-error); thus our method does not rely on a linear dependence of fluorescence intensity as a function of dye concentration, which we tested and found to hold within a narrow concentration range only. R-Actinin concentration within the tactoids was determined with the same method, using the TRITC-labeled rabbit skeletal R-actinin. The labeled R-actinin was added to F-actin both for preparation of the tactoids and for preparing a calibration sample, but in different proportions due to the same reason as described above for determining the actin concentration. Small-Angle X-ray Diffraction. The small-angle X-ray measurements were performed with the BioCAT beam line at the Argonne National Laboratory, where the X-ray beam was trimmed to a 0.5 × 1 mm spot with a brightness of 1013 photons/ s.22 The wavelength of the X-ray beam was 1.033 Å. A concentrated actin gel containing numerous micrometer-sized granules was sandwiched between two cover slides, producing a sample thickness ∼0.2 mm. To increase the diffraction signal and

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Figure 1. A phase-contrast micrograph of actin tactoids in a background solution of F-actin. The image was taken using a 40× objective lens. Most tactoids appear similar in shape, but vary in size. decrease the X-ray damage to the sample, we changed the exposure area every 3 s for a total of 25 or 36 positions. Our results showed that this method suffers much less radiation damage than exposure for a long time at a fixed position. The diffraction patterns were recorded with a high-resolution CCD camera (1024 × 1024 pixels), placed 2.39 m from the sample position. The pixel size of the camera is 0.1 mm × 0.1 mm. Thus, when a direct beam hits near one corner of the camera chip, it is capable of detecting the scattering vector q in the range of 0-2.5 nm-1. In a test experiment performed with actin bundles induced by 50 mM MgCl2, a rather sharp scattering ring was observed, with a radius corresponding to a packing spacing of 8.0 nm, which suggested tight packing of the neighboring actin filaments. In the experiments done with the tactoids, a weak scattering ring was obtained that was close to the high background scattering near the central beam. To increase the signalto-noise ratio, a computer program was written in Java, which averaged the scattering intensity along the radius of the scattering pattern. The azimuthally averaged result is shown in Figure 3B.

Results A typical experiment started by mixing substoichiometric amounts of gelsolin (1:185-500 of gelsolin/actin) and R-actinin (1:100-400 of R-actinin/actin) with column purified G-actin at a concentration of 4-8 mg/mL-1. Then 2 mM MgCl2 and 50 mM KCl were added into the protein solution. After over 1 h of incubation at room temperature for the actin polymerization, the mixture was examined under an optical microscope using the phase contrast mode to confirm that no actin bundles or granules were present. The F-actin was then concentrated by a procedure of sedimentation and resuspension as described in the materials and methods section. Incubation of the concentrated actin gel at 37 °C overnight led to the formation of large granules as shown in Figure 1. Repeated control experiments in which no R-actinin was added showed no granule formation. Addition of R-actinin at 1:50 or higher in molar ratio to actin resulted in long and thin actin bundles, the ultrastructure of which has been studied by other researchers.13,23 The abundance and the average size of granules varied depending on the preparation procedure. An extensive (22) Irving, T. C.; Maughan, D. W. In vivo X-ray diffraction of indirect flight muscle from Drosophila melanogaster. Biophys. J. 2000, 78, 25112515. (23) Meyer, R. K.; Aebi, U. Bundling of actin filaments by alphaactinin depends on its molecular length. J. Cell Biol. 1990, 110, 20132024.

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series of tests were performed in order to obtain large granules desirable for structural characterization by X-ray diffraction. The parameters that were varied include buffer components, salt concentrations, solution pH from 6.0 to 8.0, centrifugation speed and duration, and filament length, which was controlled by adding trace amounts of gelsolin (specified in Materials and Methods). The overall conclusion was that the correlation was weak between the size and shape of the granules and any of these parameters. A noticeable effect occurred when gelsolin was added in the preparation: the tactoidal granules formed by F-actin of shorter average filament length had smaller aspect ratios, thus appearing to be fatter tactoids. We also performed control experiments, making repeated preparations in order to produce actin granules without adding gelsolin into the actin/R-actinin mixture. Few granules were observed when no gelsolin was added. The mixtures of long actin filaments with even trace amounts of R-actinin tend to be very viscous prior to sedimentation. Resuspension to a larger concentration of the protein mixture frequently led to a nonuniform gel, which appeared amorphous when viewed by phase contrast optical microscopy. Therefore, gelsolin appears to be facilitating actin granule formation by reducing the entanglement of long actin filaments as they associate with R-actinin to form tactoidal aggregates. We used a trace amount of CPITC-phalloidin to detect the distribution of actin in the inhomogeneous solution containing numerous granules. The F-actin specific dye preferentially concentrated within the granules, and the concentration of actin within the granules was determined to be approximately 90 mg/mL based on the measurement of fluorescence intensity (Figure 2, image A). We added FITC-labeled gelsolin into a preparation of actin so that the gelsolin/actin ratio was 1:500, giving rise to an average filament length of 1.4 µm. The length distribution of F-actin regulated by gelsolin was characterized by negative staining electron microscopy and was shown to be exponential with average length in good agreement with that predicted by the gelsolin-actin ratio (data not shown). Ratio imaging between the gelsolin and actin fluorescence of a thin slice of giant tactoids suggested that the end distribution was uniform within the granule and that the average length of F-actin was approximately 1.7 times that of the filaments in the surrounding nematic matrix. The R-actinin distribution inside the tactoids was not uniform, with intensity variation visible at the submicrometer level (see Figure 2C). Nevertheless, the average R-actinin concentration was determined to be approximately 12-15 mg/mL using the fluorescence quantification assay described in the materials and methods section, suggesting an R-actinin/actin molar ratio of 1 to 16-20, i.e., 1 R-actinin dimer per 32-40 actin protomers within the granules. The packing of F-actin inside the granules was revealed by EM, as shown in Figure 3, for a small granule. Although the image is a two-dimensional (2-D) projection of the packing, actin filaments can be discerned. Small-angle X-ray scattering through a mixture of the actin gel containing numerous micrometer-sized granules gave intense scattering at low scattering vectors, suggesting large-scale density fluctuations. Nevertheless, a weak scattering peak was shown via radial averaging. At the range of small scattering vector q < 0.3 nm-1 the intensity falls as 1/q2, characteristic of the scattering profile for rods. This general profile is separated from the specific peak/s due to the periodic spacing as actin filaments pack into large granules, by plotting the scattering results as (I - Ib)q2 vs q (inset of Figure 3), where Ib is the background

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Figure 2. An optical slice of an actin tactoid showing the content and distribution of actin (A), gelsolin (B), and R-actinin (C). The fluorescence conjugates used were CPITC-phalloidin for F-actin, fluorescein (FITC)-gelsolin, and rhodamine (TRITC)-R-actinin, respectively. The display window for each image represents a small field of ca. 11 µm × 15 µm.

Figure 3. Structural study of F-actin/R-actinin granules: (A) An electron micrograph of a segment of a small tactoid. Individual filaments packed in the bundle, as well as defects in their packing, are discernible. (B) A small-angle X-ray scattering profile through an actin gel containing numerous tactoids. The intensity profile was azimuthally averaged (see Materials and Methods). (Inset) (I - Ib)q2 vs q plot shows a peak at q ) 0.47 nm-1, corresponding to an interfilament spacing of 15.6 nm.

scattering intensity averaged over all the q < 1.0 nm-1 data. The peak position becomes well defined in the (I - Ib)q2 vs q plot. The peak scattering vector of q ) 0.47 nm-1, identified in the inset, corresponds to an interlayer spacing of 13.4 nm. Assuming hexagonal packing of F-actin inside the tactoids, such a layer spacing translates to an interfilament spacing of approximately 15.6 nm (13.4 nm/sin(60°)). Knowing that there are 370 actin monomers per 1 µm long actin filament, this spacing corresponds to an actin concentration of 124 mg/mL. This predicted value is subject to considerable variability due to the spread of the weak peak. For instance, the peak scattering vector appears to be as low as q ) 0.36 nm-1, which would yield a larger interfilament spacing of 20.4 nm and thus a lower actin concentration of 73 mg/mL. Due to the weakness of the peak on a strong background,

we also could not rule out alternative packing geometry, such as a square lattice as proposed under different conditions,24 which would also predict a different spacing. Within the norm of these uncertainties, the actin concentration obtained from X-ray is consistent with the value of approximately 90 mg/mL, measured through the fluorescence imaging technique. Since the length of an R-actinin dimer is 32 nm, we conclude that the R-actinin cross-links must be either tilted to within 20° from the F-actin filament axis or, alternatively, the long R-actinin skips the nearest neighbors to link the ones 20-25 nm apart, or a combination of these possibilities. (24) Pelletier, O.; Pokidysheva, E.; Hirst, L. S.; Bouxsein, N.; Li, Y.; Safinya, C. R. Structure of actin cross-linked with alpha-actinin: a network of bundles. Phys. Rev. Lett. 2003, 91, 148102.

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Figure 4. Variant forms of giant actin granules. (A) A triangular granule, which appears much more rarely than tactoids but more often than any other shape. (B) Image of the same granule as (A), but at a different depth of focus. By variation of the depth of focus, the triangular granule was shown to be a flat object slightly tilted from the focal plane. (C and D) Two other variant forms of the giant actin granules.

We found during the course of our study that once large granules are formed they are stable against dilution. Some tactoids remained intact even when the sample was diluted and incubated at 37 °C over 2 days. While the American football shape is the most prevalent form of concentrated actin granules, other variants were also found in rare occurrences, such as large triangles (Figure 4) and giant tubules (Figure 5). We also observed nested granular structures, i.e., tactoids contained inside the giant tubules (Figure 5) or larger tactoids (Figure 6), which were produced by repeated cycles of sedimentation and resuspension (as described in Materials and Methods). Discussion Tactoidal granules were described originally in connection with the first-order phase transition between an orientationally isotropic phase and a nematic liquid crystalline phase of virus suspensions such as the tobacco mosaic virus (TMV).25 In the suspensions of TMV, either nematic droplets of a higher virus concentration appear as tactoids in a background of a lower concentration isotropic state or, conversely, isotropic droplets of a lower concentration appear as tactoids in a nematic background. Metastable nematic tactoids of filamentous virus (fd) have recently been observed in an isotropic mixture of fd and inert polymer dextran.26 All these observations are related to a first-order isotropic liquid crystalline phase transition, and the statistical mechanical theory underlying the phase transition has been well established.27,28 (25) Freundlich, H. Colloidal Structures in Biology. J. Phys. Chem. 1938, 41, 1151-1161. (26) Dogic, Z. Surface Freezing and a Two-Step Pathway of the Isotropic-Smectic Phase Transition in Colloidal Rods. Phys. Rev. Lett. 2003, 91, 165701-165704.

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Figure 5. Small actin tactoids embedded within a large tubule, viewed at three depths of focus. Numerous small tactoids are seen at different depths of focus, with variable orientations.

A recent theoretical work, based on minimizing the sum of surface tension and bulk elastic energy, predicts nematic droplets coexisting with an isotropic bulk solution to be of tactoidal shape of variable aspect ratios as a function of a dimensionless elastic stiffness.29 The case of actin tactoids reported here, however, differs from the case for which the theoretical prediction is applicable. The background of the actin tactoids is nematic, as we determined in an early study,30 whereas the filament concentration inside tactoids is much larger than that in the nematic state, as determined by this study. In the earlier study, however, the presence of trace amounts of the actin cross-linking protein R-actinin in the samples was not recognized. The present study not only confirms that trace amounts of R-actinin are required for inducing the formation of large granules, but also verifies that R-actinin becomes highly enriched within the tactoids, thus stabilizing the granular structures. The structure of F-actin granules proposed through this study is depicted in Figure 7. In light of the fact that R-actinin binding plays a key role in the formation of actin granules, one can alternatively imagine that the American football shape is the natural consequence of a large number of filaments added to the core of an existing actin bundle. The shape of the tactoids appears such that the surface area of these (27) Onsager, L. The effects of shape on the interaction of colloidal particles. Ann. N.Y. Acad. Sci. 1949, 51, 627-659. (28) de Gennes, P. G.; Prost, J. The Physics of Liquid Crystals; Clarendon: Oxford, 1994. (29) Prinsen, P.; van der Schoot, P. Shape and director-field transformation of tactoids. Phys. Rev. E: Stat., Nonlin., Soft Matter Phys. 2003, 68, 021701. (30) Tang, J. X.; Oldenbourg, R.; Allen, P. G.; Janmey, P. A. Tactoidal granules in concentrated actin gels: a solidlike state of protein filaments. Proceedings of the Material Research Society Fall Meeting, Symposium K: Materials Science of the Cell, 1997.

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Figure 6. Small actin tactoids embedded within two large tactoids. The relative distance and orientation changed between the two large tactoids following a slow flow of the fluid background.

granules is minimized, while in the meantime satisfying the constraint of tight packing of long actin filaments. To attain the well-defined smooth shape of these granules, a certain extent of adjustment must be in order. The thermodynamically reversible nature of proteinprotein interactions in general and the rapid on and off rates of R-actinin binding of F-actin, in particular, may provide the key molecular mechanism for such adjustment. For instance, the off-rate of R-actinin varies from on the order of 0.1-10 S-1 in the temperature range of 4-25 °C.14,15 The first-order on-rate is on the order of 1 S-1 µM-1, and it increases less steeply with temperature. The dissociation constant varies from a few tenths to several µM in the temperature range of 4-25 °C.15 These known values suggest weaker and more dynamic binding of R-actinin with increased temperature, which is expected to facilitate the formation of actin granules. Kinetics may also play a key role in the formation of the actin granules related to the specific structure of R-actinin. R-Actinin is known to exist as dimers, with actin binding sites near both ends of each dimer. Although the simultaneous binding of two neighboring filaments, i.e., crosslinking, lowers the free energy, bringing actin filaments closer than their average spacing in solution results in a loss of entropy. Therefore, at low R-actinin concentrations, the granular aggregates are energetically favored only when the actin concentration is sufficiently high and thus the entropic loss is overcompensated by the energy gain due to the cross-linking. Furthermore, since the binding only occurs when the proteins collide or at least are within sub-nanometer proximity, formation of actin/R-actinin granules necessarily involves crossing a free energy barrier and thus is kinetically disfavored. Additionally, in the

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Figure 7. Proposed structure of an actin tactoid (A) and a triangular granule (B) embedded in a nematic actin gel. The packing of F-actin within the granule is depicted with R-actinin dimers as cross-linkers (red). Gelsolin, which is predictably bound to the barbed end of each F-actin, is not shown in the simple drawing.

absence of R-actinin, the negatively charged actin filaments tend to repel each other at close approach under the ionic conditions of this study, thus contributing to the energy barrier which prohibits the formation of actin bundles containing tightly packed filaments.9,31 The kinetic barrier described above may account for the observed irreversible nature of tactoids upon dilution. Actin granules containing R-actinin may be energetically favored even at low initial concentrations; their formation is practically inhibited by a kinetic barrier. On the basis of our experimental observation, once actin concentration is increased to above 20 mg/mL, the entropic penalty for aggregation is sufficiently reduced so that the kinetic barrier is obliterated. Consequently, tactoidal granules form in concentrated actin samples containing trace amounts of R-actinin. Once the tactoids are formed, the attractive interaction mediated by R-actinin between neighboring actin filaments becomes strong enough to hold the packed assembly together. Thus, the tactoidal granules are held together upon dilution, even to a concentration lower than that initially required for their formation. One alternative explanation for the irreversible nature of the tactoids is that the protein is denatured within the large granules. However, our observation of certain features tends to contradict this suggestion. For example, the phalloidin stabilization of F-actin did not appear to affect the formation of tactoids, and the tactoids were formed readily from actin filaments with or without phalloidin bound. It is therefore most likely that the tactoids consist of intact actin filaments. Note also that the binding of phalloidin to F-actin is highly specific, which involves contacts with three adjacent monomers along (31) Tang, J. X.; Ito, T.; Tao, T.; Traub, P.; Janmey, P. A. Opposite Effects of Electrostatics and Steric Exclusion on Bundle Formation by F-actin and Other Filamentous Polyelectrolytes. Biochemistry 1997, 36, 12600-12607.

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the actin filament.32 We found that TRITC-labeled phalloidin molecules label entire tactoids over the time course of several minutes, supporting the suggestion that actin inside the tactoids remains as intact filaments. Such an observation also confirms our model of loose packing of F-actin within the tactoids (15.6 nm axial spacing in comparison with the 8 nm actin diameter), leaving sufficient space accessible for solvent and other small molecules. The proposed structure of the R-actinin and F-actin complex inside the tactoidal granules is notably different from predictions based on a recent X-ray study of mixtures of F-actin and R-actinin.24 In the latter study, a square lattice is proposed with an interfilament distance nearly the length of R-actinin. Large distortions are also suggested based on the X-ray data to establish the square lattice model. The X-ray peak position we observed is different, perhaps due to the different preparation procedures used. Despite a smaller molar ratio of R-actinin/ actin in our initial solution (1:200), the self-assembled granules contain a much larger proportion of R-actinin (between 1:16 and 1:20 in molar ratio of actin/R-actinin), and a much tighter spacing of approximately 16 nm between actin filaments within the dense granules is obtained. These results are consistent with the concentrations of both proteins determined independently by the fluorescent techniques in this study. The two different structures proposed above are relevant to certain actin/R-actinin structures in vivo. Inside both the z-disk and focal adhesion complex,1 the spacing between the neighboring actin filaments is large and comparable to the length of R-actinin, with enough space to accommodate intact myosin and potentially allow direct acto-myosin interaction. The relatively tight packing of F-actin within the tactoids is similar to certain physiologically aberrant occurrences such as the rod body33 and the Hirano body.34,35 In addition to actin and R-actinin, there are a number of other actin binding proteins that occur in these granular structures in the more complex

cellular and pathological settings, such as ADF/cofilin.36,37 In disease states such as nemaline myopathy,38 it appears that a certain imbalance or loss of specific structural regulation gives rise to the formation of similar granular structures, which is mimicked by the simplified model complex we characterized in vitro. In summary, we discovered the formation of large granules of F-actin, facilitated by the common crosslinking protein R-actinin and the filament length regulating protein gelsolin. We propose loose parallel packing of actin filaments inside the tactoidal granules and a slight local deviation from the parallel packing in large variant structures such as triangles and tubules. We propose a qualitative free energy analysis to explain the mechanism of granular formation. We also suggest a kinetic mechanism for the stability of the granules against dilution. Further study on the molecular level interactions is needed in order to understand the packing of the actin filaments, as well as how a variety of granules with distinct shapes are formed.

(32) Allen, P. G.; Janmey, P. A. Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin. J. Biol. Chem. 1994, 269, 32916-32923. (33) Tangkawattana, P.; Karkoura, A.; Muto, M.; Yamano, S.; Taniyama, H.; Yamaguchi, M. Cardiac rod body: hypertrophic Z-line in an aged pony. Acta Anat. 1996, 155, 266-273. (34) Galloway, P. G.; Perry, G.; Gambetti, P. Hirano body filaments contain actin and actin-associated proteins. J. Neuropathol. Exp. Neurol. 1987, 46, 185-199. (35) Maselli, A. G.; Davis, R.; Furukawa, R.; Fechheimer, M. Formation of Hirano bodies in Dictyostelium and mammalian cells induced by expression of a modified form of an actin-cross-linking protein. J. Cell Sci. 2002, 115, 1939-49.

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Acknowledgment. We thank Phil Allen (Harvard Medical School), Paul Janmey (University of Penn.), and Josef Ka¨s (University of Leipzig), who were involved in the preliminary phase of this study and who continued to offer materials and suggestions. We thank Dr. Bridget Carragher and Amy Reilein of the Imaging Technology Group at the Beckman Institute, UIUC, for taking the electron micrograph (Figure 3A) and Dr. Barry Stein of Indiana University for kind assistance with confocal microscopy. The X-ray image was taken at the Advanced Photon Source (APS), Argonne National Laboratory (ANL), with kind help from Dr. Thomas Irving using the BioCAT beamline. We also thank Ms Liang Zhong for helping to write a radial averaging program used for analysis of the X-ray data. This work was supported by NSF DMR-9988389, DMR-0405156, and NIH R01 HL67286.

(36) Minamide, L. S.; Striegl, A. M.; Boyle, J. A.; Meberg, P. J.; Bamburg, J. R. Neurodegenerative stimuli induce persistent ADF/cofilinactin rods that disrupt distal neurite function. Nat. Cell Biol. 2000, 2, 628-636. (37) Ono, S.; Abe, H.; Nagaoka, R.; Obinata, T. Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells. J. Muscle Res. Cell Motil. 1993, 14, 195-204. (38) Laing, N. G.; Wilton, S. D.; Akkari, P. A.; Dorosz, S.; Boundy, K.; Kneebone, C.; Blumbergs, P.; White, S.; Watkins, H.; Love, D. R., et al. A mutation in the alpha tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy. Nat. Genet. 1995, 9, 75-79.