Observation of the Three-Dimensional Structure of Actin Bundles

Dec 29, 2007 - Hokkaido University, Sapporo 060-0814, Japan. Received September 24, 2007; Revised Manuscript Received November 8, 2007...
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Biomacromolecules 2008, 9, 537–542

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Observation of the Three-Dimensional Structure of Actin Bundles Formed with Polycations Kazuhiro Shikinaka, Hyuckjoon Kwon, Akira Kakugo, Hidemitsu Furukawa, Yoshihito Osada,† and Jian Ping Gong* Department of Biological Science, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan

Yoshitaka Aoyama,‡ Hideo Nishioka,‡ and Hiroshi Jinnai§ JEOL Ltd., Akishima 151-0063, Japan, and Department of Polymer Science and Engineering, Kyoto Institute of Technology, Kyoto 606-8585, Japan

Takaharu Okajima Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University, Sapporo 060-0814, Japan Received September 24, 2007; Revised Manuscript Received November 8, 2007

Three-dimensional structures of actin bundles formed with polycations were observed by using transmission electron microtomography and atomic force microscopy. We found, for the first time, that the cross-sectional morphology of actin bundles depends on the polycation species and ionic strength, while it is insensitive to the degree of polymerization and concentration of polycation. Actin bundles formed with poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary show a ribbon-like cross-sectional morphology in low salt concentrations that changes to cylindrical cross-sectional morphology with hexagonal packing of the actin filaments in high salt concentrations. Contrastingly, actin bundles formed with poly-L-lysine show triangular cross-sectional morphology with hexagonal packing of the actin filaments. These variations in cross-sectional morphology are discussed in terms of anisotropy in the electrostatic energy barrier.

Introduction Actin, which is an abundant protein found in all eukaryotic cells, not only provides the cell with mechanical strength but also participates in cell movements such as cytokinesis, cell crawling, cytoplasmic streaming, and muscle contraction.1 In physiological conditions, globular actin (G-actin) monomers are self-assembled into linear filaments (F-actin). F-Actins also assemble into parallel bundles or form a cross-linked network in the presence of actin linker proteins. It is believed that specific linker proteins are responsible for the morphology of actin assemblies.2 However, it has also been reported that the various morphologies of actin bundles such as Drosophila bristle, nurse cell strut, stereocilia, and the acrosomal process are independent of the specific type of linker proteins.3 Some linker proteins such as R-actinin and fascin show both bundling and crosslinking activities.4–6 These facts indicate that the morphology of actin assemblies is determined not only by specific linker proteins but also by other factors such as the concentration of the linker proteins, environmental conditions (ionic strength or pH), and the kinetics of actin-linker protein interactions when the cell is in a state of nonequilibrium. Although many studies on the cellular mechanism of actin organization have been performed, the dominant factor determining the morphology and size of actin architecture remains poorly understood. * Corresponding author: [email protected]. † Present address: Riken, Saitama 351-0198, Japan. ‡ JEOL Ltd. § Kyoto Institute of Technology.

F-Actin, carrying negative charges, behaves as a rodlike molecule with a persistence length of ∼10 µm7 and forms large bundles with various polycations in vitro.4,8 The growth and structure of actin bundles formed with multivalent cations have been studied by many researchers.9–11 We have investigated actin bundle formation with various polycations and found that F-actins form bundles above a critical polycation concentration. The binding between negatively charged F-actins is originated from condensation of polycation to F-actins, which is entropically driven by counterion release from both F-actin and polycation. The larger the degree of polymerization, N, the larger the entropy gain by binding. The critical polycation concentration for inducing bundle formation (CPc) decreases dramatically with an increase in N. We further found that the growth in bundle thickness D is independent of that in bundle length L.12–14 Our studies have shown that the increase in the D of actin bundles is nearly complete in the initial stages of bundle growth, while a dramatic increase in L starts later, after the completion of the increase in D. Additionally, we found that D decreases with an increase in the polycation-mediated attraction between F-actins but is hardly influenced by the actin concentration (CA); however, L increases with increasing CA. It has been also reported that in the case of actin bundles formed with Mg2+, the longitudinal growth started after the lateral growth, the same with the polycation system9. On the basis of these results, we have proposed an anisotropic nucleation-growth model in which the D of the actin bundles is determined by the critical nucleus size, whereas the L of the actin bundles is determined by the concentration of free F-actins relative to nucleus concentration.15

10.1021/bm701068n CCC: $40.75  2008 American Chemical Society Published on Web 12/29/2007

538 Biomacromolecules, Vol. 9, No. 2, 2008 Chart 1. Molecular Structure of Poly-N-[3-(dimethylamino)propyl] Acrylamide Methyl Chloride Quaternary (PDMAPAA-Q) (a) and Poly-L-lysine (b)

Once nuclei of critical thickness are formed, an increase in L is thermodynamically favored; that is, the increases in D and L both favor a gain in entropy. However, the electrostatic repulsion of the side-by-side assembly of F-actins is stronger than that of their end-to-end annealing owing to the rodlike polyelectrolyte nature of F-actin. Therefore, the increase in D is kinetically suppressed, while the increase in L is kinetically favored in the subsequent growth process. In the above study, the D was determined using transmission electron microscopy (TEM) projection images, which smear the cross-sectional structure information. The cross-sectional structures of actin bundles, which might affect their bending rigidity,16 have been studied using the scatter approach.17,18 However, no information has been reported by real-space observation. In this paper, we observe the cross-sectional morphology of the actin bundles formed with poly-N-[3(dimethylamino)propyl] acrylamide methyl chloride quaternary (PDMAPAA-Q) or poly-lysine (p-Lys) in various salt and polymer concentrations. Transmission electron microtomography (TEMT)19,20 and atomic force microscopy (AFM) were used for observing the three-dimensional structure of actin bundles. Various types of anisotropic cross-sectional morphologies have been observed depending on the polycation species and salt concentration. These results will provide new insight into the mechanism of actin bundle formation.

Experimental Procedures Sample Preparations. Actin Preparation. G-Actin was purified from scallops by using the method of Spudich et al.21 Fluorescently labeled filamentous actin (later denoted as F-actin) was obtained by stoichiometrically mixing G-actins and rhodamine-phalloidin (Molecular Probes No. 4171) in F-buffer (5 mM HEPES (pH 7.2), 0.2 mM ATP, 0.2 mM CaCl2, 0.1 M KCl, 2 mM MgCl2) for 24 h at 4 °C. Phallodin binds to G-actin stoichiometrically and stabilizes F-actin against depolymerization at a low concentration of G-actin. Polycation Preparation. Poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary (PDMAPAA-Q; Chart 1a) was prepared by the radical polymerization of a 1.0 M aqueous solution of the DMAPAA-Q monomer (Tokyo Kasei Co., Ltd.) in the presence of 0.2 mol % potassium persulfate (Tokyo Kasei Co., Ltd.). Poly-L-lysine (p-Lys, Sigma; Chart 1b) of various degrees of polymerization (N) was used as purchased. PDMAPAA-Q and p-Lys were diluted with HEPES buffer (5 mM HEPES (pH 7.2), 0.2 mM ATP, 0.2 mM CaCl2, 2 mM MgCl2) and then used for experiments. Actin Bundle Preparation. Actin bundles were obtained as in our previous paper.14,15 The bundles were prepared by mixing F-actin solution (1 µL; 0.1 mg/mL) with polycation and KCl solution (9 µL) to yield a 10 µL reaction mixture. The actin concentration CA, expressed in terms of G-actin concentration, was kept constant at 0.01 mg/mL (corresponding to 2.32 × 10-7 M). The PDMAPAA-Q or p-Lys concentration CP, expressed in terms of cation monomer units, was varied from 10-6 to 10-1 M. The KCl concentration CS ranged from 0.01 to 0.3 M. The samples were incubated at room temperature for 60 min before observation by TEMT, AFM, and fluorescence microscopy.

Shikinaka et al. Measurement. Transmission Electron Microtomography. A tilt series of TEM projections was obtained typically from -60° to 60° in 1° increments using a JEM-2200FS (JEOL Ltd., Japan) operated at 200 kV. A drop of actin bundle solution of approximately 10 µL was put on carbon-coated 400-mesh copper grids (H100, Veco, Co. Ltd., Holland), which were coated with a Formvar film and were rendered hydrophilically by glow discharge in a reduced pressure by using ion sputter (E-1010, Hitachi Ltd.). After adsorption was allowed for 3 min, the grids were stained with 1 drop of 2% (w/v) uranyl acetate (pH 4.0), then the grids were air-dried. The digital data of the transmitted images were collected with a slow-scan CCD camera (Gatan USC1000, Gatan Inc.). The magnification of the TEM was 50000× for each specimen. A projection at each tilt angle was acquired with a frame size of 512 × 512 pixels. To obtain achromatic projections, only the transmitted and elastically scattered electrons (electron energy loss 0 (30 eV) were selected by an energy filter installed in the JEM-2200FS (Omega filter, JEOL Ltd., Japan). Prior to the transmission electron microtomographic observations, we deposited 5 nm diameter gold particles (GCN005, BBInternational Ltd., U.K.) from an aqueous suspension for use as fiducial markers for observing the alignment of the tilt series of the TEM images. The tilt series of the TEM images was carefully aligned by the fiducial marker method and then reconstructed on the basis of the filtered-back-projection method.20 TEMT measurement was performed only one to three times for each sample due to difficulty in the measurement. Atomic Force Microscopy. A drop of actin bundle solution of approximately 200 µL was put on the surface of a freshly cleaved mica piece (1 cm2). After adsorption was allowed for 30 min, AFM observation was performed using an MFD-3D (Asylum Research. Co., Santa Barbara, CA) operated in the amplitude-modulation mode in HEPES buffer at room temperature.22 Imaging was conducted using triangular Si3N4 cantilevers with a nominal spring constant of 0.15 N/m and a tip diameter of 40 nm (OMCL-TR800PSA, Olympus, Japan). Fluorescence Microscopy. A cover glass was placed on a slide glass equipped with two spacers (∼0.2 mm high) on both sides to form a cell. The reaction mixture (approximately 10 µL) was introduced into the cell by a micropipet. Then, the cell was placed on the stage of a fluorescence microscope (Olympus BX 50) and observed under a 60× objective lens. The fluorescence images were recorded by a CCDcamera (Olympus CD-300T-RC).

Results and Discussion Figure 1 shows the fluorescent and TEMT images of actin bundles formed with PDMAPAA-Q at various CS and CP. Planes parallel to the specimen grids are denoted as the x-y plane, where the x axis was chosen as the direction parallel to the bundle length. As a guide, schematic views of F-actin are also shown in the figure. In the absence of polycations (CP ) 0 M), F-actins at a concentration of 0.01 mg/mL are present as single filaments of thickness ∼10 nm; the filaments exhibit a polydisperse length distribution of 1-10 µm, with an average of approximately 5 µm.15 There is no bundle formation even in the presence of divalent cations due to the low concentration of the divalent cations in the present experiment condition. The actin bundles show a ribbon-like cross-sectional morphology with lateral packing of the actin filaments in the presence of polycations at a low CS (CP ) 10-5 M, 10-1 M and CS ) 0.01 M). The cross-sectional morphology changes to cylindrical with hexagonal packing at a high CS (CP ) 10-5 M and CS ) 0.3 M). The width and height of the bundles formed at various CP and CS are summarized in Figure 2. At a constant CS of 0.01 M, the width increases with an increase in CP, while the height barely depends on the CP, showing a value that is the same as that of F-actin (circular symbols in Figure 2). In contrast, at a constant CP of 10-5 M, both the width and height increase with an increase in CS from 0.01 to 0.3 M (triangular symbols in

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Figure 1. Fluorescence images and cross-sectional images of the actin bundles formed with PDMAPAA-Q at various concentrations of polymer (CP) and KCl (CS) obtained by fluorescence microscopy and TEMT. Arrows along the x-y plane of the TEMT images represent the positions of the images taken along the y-z plane. The fluorescence and cross-sectional images presented are drawn to scales of 10 µm and 50 nm, respectively.

Figure 2). This result indicates that at a low CS, the lateral growth of nuclei is anisotropic; contrastingly, it becomes isotropic when the CS is high. In a previous study, the D was determined from TEM projection images, which smear the crosssectional information.14 From the present TEMT observation, we know that the D observed by TEM is related to the bundle width estimated by TEMT observation, and we found that the D value and the bundle width are well-related in their dependence on the values of CP and CS. However, since the anisotropic morphology of the actin bundles might have been artificially induced during the preparation of the TEM specimens, we also observed the actin bundles by AFM in the liquid state (Figure 3). The AFM images reveal that the height increases with an increase in CS from 0.01 to 0.3 M at a constant CP of 10-5 M, which corresponds well with the results obtained by TEMT observation. The width elucidated by AFM was approximately three times greater than that observed by TEMT. We consider that the increased width observed by AFM is due to the diameter of the cantilever tip that was 40 nm, comparable with the size of the Actin bundles (∼45 nm). Considering this

limitation in spatial resolution, we assume that the results from TEMT and AFM are in good agreement. The fluorescent images in Figure 1 revealed that the actin bundles undergo morphological changes from a compact globule structure in which the actin bundles cross over (CP ) 10-5 M and CS ) 0.01 M; the average long axis of globule is 17.4 µm as shown in ref 14) to an extended structure in which the actin bundles are straight (CP ) 10-5 M and CS ) 0.3 M; the average long axis of bundle is 73.7 µm as shown in ref 14) on a micrometer scale, which is the same as revealed by a previous report.14 On the basis of Figures 1 and 3, these morphological changes might be attributed to the cross-sectional changes in the structure of actin bundles from a ribbon-like structure that is flexible (CP ) 10-5 M and CS ) 0.01 M) to a cylindrical structure that is rigid (CP ) 10-5 M and CS ) 0.3 M) on a nanometer scale. In the previous study, by florescent image and TEM, we found that the actin bundles form thick but short bundles when increases in CP while keeping the CS low (CP ) 10-1 M and CS ) 0.01 M, the average long axis of bundle is 6.48 µm as shown in ref 14). Therefore, we have assumed,

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Figure 2. The width (closed symbols) and height (open symbols) of the actin bundles formed at various PDMAPAA-Q concentrations (CP) and KCl contentrations (CS). (b, O), CS ) 0.01 M; (2, 4), CS ) 0.3 M. The width and height, defined in the illustration, are measured along the guidelines in the schematic views of Figure 1. The size of the bundle formed at CP ) 10-5 M, CS ) 0.01 M, and CP ) 10-1 M, CS ) 0.01 M, was the average of two and three samples, respectively. Other data were measured for one sample. The diameter of F-actin is 11.8 nm, as determined from Figure 1, which corresponds well with our previous result.13 The relative size to the F-actin diameter, which means the ratio of the bundle width or height to the F-actin diameter, is also displayed in the figure as the right vertical axis.

which is not correct, that the actin bundle prepared in this condition has the same cross-sectional radius with that prepared in a high ionic strength (CP ) 10-5 M and CS ) 0.3 M). However, we found from the TEMT observation that actin bundles prepared in CP ) 10-1 M and CS ) 0.01 M show the ribbon-like structure, which is different from the cylindrical structure prepared at CP ) 10-5 M and CS ) 0.3 M. So the CP-CS morphology diagram as shown in Figure 1 of ref 14 should be modified. The electrostatic attraction between negatively charged F-actin and positively charged polyelectrolyte increases with the increase in the degree of polymerization N at a constant ionic strength because of the favored gain in entropy at a large N. In our previous work, we studied the effect of N on bundle formation using p-Lys. We found that the critical p-Lys concentration for inducing bundle formation (CPc) decreases dramatically with the increase in the N of the p-Lys; CPc at N ) 3, 5, 26, 1336 is 7.5 × 10-3 M, 5.0 × 10-5 M, 5.0 × 10-6 M, and 3.3 × 10-7 M, respectively, where CPc is expressed in terms of the concentration of Lys monomer units.15 Using twodimensional TEM, we revealed that the D of the actin bundles formed just above CPc decreased with an increase in N, showing values of 59.2 ( 18.2, 41.6 ( 12.7, 33.5 ( 9.4, and 21.4 ( 5.5 nm for N ) 3, 5, 26, and 1336, respectively. This behavior can be understood by the nucleation mechanism; that is, the stronger the attraction between F-actins mediated by polycations, the thinner the bundles formed. Here, we observe the cross-sectional morphology of the p-Lys system. Figure 4 shows TEMT and AFM images of the actin bundles formed with p-Lys of various N values at their CPc, while maintaining the CS at a constant of 0.01 M. As shown in Figure 4 (y-z plane), the bundles show a triangular cross section with hexagonally arranged F-actin filaments, regardless of substantial changes in the N of the p-Lys. Further, the width of the actin bundles decreases with an increase in the N or CP of

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the p-Lys, while the height of bundles barely depends on the N or CP of the p-Lys (Figure 5). As discussed above, the D as determined from the TEM projection images is related to the width of the actin bundles determined by TEMT. Therefore, we considered that the decrease in the width of the actin bundles with an increase in the N is in agreement with a previous result that showed a decrease in the D with an increase in the N. Rodlike rigid polyelectrolyte aggregation has been studied for different biopolymers.8–11,17,18,23–31 In these studies, it has been reported that the hydrated sizes of the multivalent cation influence its function as aggregating agent of negatively charged rodlike polyelectrolytes.10,23 The pointlike ions are assumed to neutralize the charge of rodlike polyelectrolyte completely, while the large ions have steric hindrance in the packing, which prevents charge neutralization of polyelectrolyte.24 It has also been reported that a slight difference in the surface charge density of negatively charged rodlike polyelectrolyte influences its condensation with positively charged multivalent ions.25 The different structure of actin bundles formed with PDMAPAA-Q and p-Lys seems in common with these results; that is, the difference in the chemical structure of monomer unit of polycation may influence the packing of F-actins and, therefore, influence the cross-sectional structure of the actin bundle. From the experimental results, we know the following: (1) The cross-sectional morphology of actin bundles (ribbonlike or triangular) is sensitive to the chemical structure of polycation, while it is not sensitive to the length of polycation; i.e., when we change the length of p-Lys, the basic morphology (triangle-like with hexagonal packing) does not change. This suggests that a difference in the binding specificity of polymers to F-actin influences the morphology of the actin bundles. (2) The anisotropic lateral growth of actin bundles has an electrostatic origin; i.e., the cross-sectional morphology becomes isotropic with increase in the KCl concentration, due to a screening of electrostatic interaction. (3) The kinetics of bundle formation may play a role in the anisotropic growth; i.e. at higher CP, the width of the ribbon and the triangle increases, while the height does not change, therefore, the anisotropy of cross-sectional morphology increases. At present, we have no satisfactory explanation on how this anisotropy is induced. According to the anisotropic nucleationgrowth model, which states that D is determined at the nucleation stage, the anisotropy in the lateral direction should occur in the initial nucleation stage when F-actins form nuclei mediated by polycations.15 We try to discuss the origin of anisotropy from several points. One is the anisotropy in the structure of G-actin. This does not appear to be the case since F-actin behaves like an isotropically negatively charged rod because the nonhomogeneous charge distribution on the surface of G-actin (several nanometers) is spatially much less than the length of F-actin (several micrometers) that comprises a double helical structure of G-actins with a helical pitch of 36 nm. The other is the long-range interaction between charged rods. We place the first F-actin in the x-y plane, aligned in the x direction (refer to Figure 6). When a second free F-actin approaches the first one, the two F-actins bind due to the mediation of polycations (not shown in Figure 6). We locate the two bound F-actins, for example, in the x-y plane. When a third free F-actin approaches the two bound actins, the energy barrier of electrostatic repulsion in the z direction is higher than that in the y direction. This anisotropic energy barrier induces favorable binding of F-actin from the y direction. With the increase in anisotropy, a difference in the binding energy barrier

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Figure 3. AFM images and height profiles of the actin bundles formed with PDMAPAA-Q in (a) CS ) 0.01 M, (b) CS ) 0.3 M at a constant CP of 10-5 M. Height profiles were considered along the dashed line in the images.

Figure 4. TEMT, AFM images, and height profiles according to the AFM image of the actin bundles formed with p-Lys of various degrees of polymerization N. The actin bundle formation was performed at a CP just above the critical concentration of the corresponding N; CP ) 10-2 M (N ) 3), 10-5 M (N ) 26), 10-6 M (N ) 1338), and the CS was kept constant at 0.01 M. Scale bars represent 50 nm. Illustration on the right of the cross-sectional images of N ) 26 is a schematic view of the TEMT image. The AFM image depicts the actin bundle formed with p-Lys at N ) 26.

between the y direction and z direction increases; this favors the growth of nuclei with a ribbon-like structure. Thus, lateral growth is kinetically favored but height growth is suppressed during the formation of actin bundles. Ribbon-like nuclei larger than the critical nuclei size start longitudinal growth in the x axis direction. As a result, actin bundles with the ribbon-like cross section are formed. Increase in CS screens the electrostatic repulsion between the nuclei and F-actin in the z direction, leading to isotropic binding to form nuclei with a cylindrical cross section of hexagonal packing at high CS. The triangular structure of actin bundles formed with p-Lys may be associated with a smaller difference in the binding energy barrier between the y and z direction. The enhanced

anisotropy at a high CP for the p-Lys system may be originated from the relaxation of the binding energy barrier, that is, the high binding energy barrier in the z direction may be lowered by relaxation, thus favoring binding from the z direction in a slow binding process at a low CP, while it is forbidden at a high CP.

Conclusions TEMT and AFM observations reveal that the actin bundles have anisotropic cross-sectional structure, which depends on the polymer species, the polymer concentration CP, and the KCl concentration CS. The actin bundles formed with PDMAPAA-Q

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of actin bundles might be biologically essential for cellular activity. A quantitative relationship between the cross-sectional structure of actin bundles and its mechanical properties will be discussed in a separate paper. Acknowledgment. This research is financially supported by SORST, JST, and the Ministry of Education, Science, Sports, and Culture, Japan (Grant-in-Aid of Specially Promoted Scientific Research).

References and Notes

Figure 5. The width (closed circles) and height (open circles) of the actin bundles formed with p-Lys at various N at CP ) 10-2 M (N ) 3), 10-5 M (N ) 26), 10-6 M (N ) 1338), and CS ) 0.01 M. Each data set was measured for one sample. The dashed curves are drawn for guidance.

Figure 6. Schematic illustration of anisotropic nuclei formation of F-actin bundles due to electrostatic repulsion. After two actins bound together in the y direction, the binding of the third F-actin occurs from the y direction (a) and has a lower energy barrier than if it occurs from the z direction (b). Therefore, the binding of the third F-actin from the y direction is favored. With the increase in anisotropy, the difference in the binding energy barrier between the y direction and z direction increases, and this favors the growth of nuclei with a ribbon-like morphology.

show a ribbon-like cross-sectional morphology with monolayer packing at a low CS, while the width of the ribbon increases with CP, and a cylindrical cross-sectional morphology with hexagonal packing at a high CS. On the other hand, actin bundles formed with p-Lys show a triangular cross-section morphology with hexagonal packing, irrespective of the degree of polymerization N and CP of p-Lys. The anisotropy in the crosssectional structure increases with the polymer concentration CP. The anisotropy in the cross-sectional structure of actin bundles may originate from an anisotropic electrostatic interaction between F-actins. In cells, actin filaments are organized into bundles that have various cross-sectional morphologies,32,33 and these bundles support and stabilize cellular protrusions and invaginations.34 It is considered that the cross-sectional structure of an actin bundle influences its mechanical properties, such as bending rigidity.16 For example, if a bundle has a noncircular cross section, its bending rigidity will depend on the direction of the bending. Therefore, the difference in the cross-sectional structure

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