Mechanism on Polarity Sorting of Actin Bundles Formed with Polycations

Dec 30, 2008 - In this paper we explored factors that determine the polarity of an Actin bundle formed with polycation through electrostatic interacti...
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Langmuir 2009, 25, 1554-1557

Mechanism on Polarity Sorting of Actin Bundles Formed with Polycations Kazuhiro Shikinaka,† Akira Kakugo, Yoshihito Osada,‡ and Jian Ping Gong* Department of Life Sciences, Graduate School of Science, Hokkaido UniVersity, Sapporo 060-0810, Japan ReceiVed July 1, 2008. ReVised Manuscript ReceiVed NoVember 16, 2008 In this paper we explored factors that determine the polarity of an Actin bundle formed with polycation through electrostatic interaction. We found that the polarity decreases with an increase in the polycation concentration while it hardly depends on the KCl salt concentration. Additionally, the polarity of the Actin bundle increases with an increase in the degree of polymerization of the polycation at a constant polymer concentration. From these results we proposed that the kinetics of nuclei formation determines the polarity of the Actin bundle.

Introduction Actin is a ubiquitous protein which is found in many living organisms and plays an important role in numerous vital activities, such as cell motility,1 cytokinesis,2 phagocytosis,3 structural support,4 and contraction of myofibrils. The polymorphism of Actin assembles formed from linear filament of Actins with helical structure (F-Actin) and cations (K+, Mg2+)5 or Actin linker proteins is responsible for such multifunctionalities.6-8 For achieving the cell motility and muscle contraction F-Actins in the assemble have a preferential polarity.9-14 It has been reported that the polarity is influenced by the specific type of Actin linker proteins. For example, Actin bundles formed with fascin15 or P-135-ABP16 have a uniform polarity, while bundle formed with R-actinin has a random polarity.17 However, the mechanism of how the polarity is sorted has been poorly understood. Elucidation of this mechanism is important for understanding the cell activity and muscle contraction. Recently, we reported that Actin bundles are obtained when F-Actin is mixed with some synthetic polymers carrying positive charges.18 The Actin bundles show a sliding motion on the surface * To whom correspondence should be addressed. E-mail: gong@ sci.hokudai.ac.jp. † Present address: Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Koganei 184-8588, Japan. ‡ Present address: Riken, Saitama 351-0198, Japan.

(1) Cooper, J. A. Annu. ReV. Physiol. 1991, 53, 585–605. (2) Umeda, M.; Emoto, K. Chem. Phys. Lipids 1999, 101(1), 81–91. (3) Chimini, G.; Chavrier, P. Nat. Cell Biol. 2000, 2, E191. (4) Paula, K. Y.; Dao-Yi, Y.; Valerie, A. A.; Ulrich, S.; Er-Ning, S.; Stephen, J. C. Exp. Eye Res. 1997, 65(3), 379–389. (5) Selden, L. A.; Gershman, L. C.; Estes, J. E. J. Muscle Res. Cell Motil. 1986, 7(3), 215–224. (6) Machesky, L. M.; Insall, R. H. Curr. Biol. 1998, 8(25), 1347–1356. (7) Svitkina, T. M.; Borisy, G. G. J. Cell. Biol. 1999, 145, 1009–1026. (8) Kureishy, N.; Sapountzi, V.; Prag, S.; Anilkumar, N.; Adams, J. C. Bioessays 2002, 24(4), 350–361. (9) Howard, J. Mechanics of motor proteins and the cytoskeleton; Sinauer Associates Inc.: Sunderland, MA, 2001; pp 119-134. (10) Katoh, K.; Hammer, K.; Smith, P. J. S.; Oldenbourg, R. Mol. Biol. Cell 1999, 10, 197–210. (11) Lin, C. H.; Espreafico, E. M.; Mooseker, M. S.; Forscher, P. Neuron 1996, 16, 769–782. (12) Mallavarapu, A.; Mitchison, T. J. Cell Biol. 1999, 146, 1097–1106. (13) Okabe, S.; Hirokawa, N. J. Neurosci. 1991, 11, 1918–1929. (14) Lewis, A. K.; Bridgman, P. C. J. Cell Biol. 1992, 119, 1219–1243. (15) Ishikawa, R.; Sakamoto, T.; Ando, T.; Higashi-Fujime, S.; Kohama, K. J. Neurochem. 2003, 87(3), 676–685. (16) Yokota, E.; Shimmen, T. Planta 1999, 209, 264–266. (17) Djinovic-Carugo, K.; Young, P.; Gautel, M.; Saraste, M. Cell 1999, 98, 537–546. (18) Kakugo, A.; Shikinaka, K.; Matsumoto, K.; Gong, J. P.; Osada, Y. Bioconjugate Chem. 2003, 14(6), 1185–1190.

coated with myosin by coupling to ATP hydrolysis19 as seen in F-Actin,20-22 and the sliding velocity of these bundles depends on the species of polycation.23 We found that in the bundle the polarity of individual F-Actin was preserved while the F-Actins were aligned in parallel or antiparallel structure. As a result, the global polarity of the Actin bundle depends on the polycation species. We further found that there was a linear relationship between the polarity of the Actin bundle and its sliding velocity.23 Thus, the polarity of the Actin bundle also plays an essential role in its in vitro motility. In our previous study an anisotropic nucleation-growth mechanism for Actin bundle formation was proposed based on the following facts: (1) the cross-sectional growth of Actin bundles is nearly complete in the initial stages of bundle growth, while a dramatic length growth starts later, and (2) the bundle thickness D decreases with increasing polymerization degree of the polycation (N) but is less influenced by the Actin concentration (CA), while the bundle length increases with increasing CA.24 A similar growth mechanism has also been reported by other researchers.25-27 In this mechanism the cross-sectional shape of the Actin bundles is determined in the nucleation stage of bundle growth. Therefore, it can be predicted that the polarity of the Actin bundle is formed in the nucleation stage. In this paper we explore the factors to determine the polarity of the Actin bundle by observing the nuclei formation process. We use poly-L-lysine of various degree of polymerization N as the polycation. As an experimental result the polarity of Actin bundles depends not on salt concentration (CS) but on polycation concentration (CP). Moreover, we found that the polarity also depends on the value of N. These results suggest that the polarity is affected not by the strength of polycation-mediated attraction between Actins but by the kinetics of nuclei formation of Actin bundles. (19) Kakugo, A.; Sugimoto, S.; Gong, J. P.; Osada, Y. AdV. Mater. 2002, 14(16), 1124–1126. (20) Uyeda, T. Q. P.; Warruch, H. M.; Kron, S. J.; Spudich, J. A. Nature 1991, 352, 307–311. (21) Huxley, H. E. J. Biol. Chem. 1990, 265, 8347–8350. (22) Korn, S. J.; Spudich, J. A Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6272– 6276. (23) Kakugo, A.; Shikinaka, K.; Takekawa, N.; Sugimoto, S.; Osada, Y.; Gong, J. P. Biomacromolecules 2005, 6, 845–849. (24) Kwon, H. J.; Tanaka, Y.; Kakugo, A.; Shikinaka, K.; Furukawa, H.; Osada, Y.; Gong, J. P. Biochemistry 2006, 45(34), 10313–10318. (25) Haviv, L.; Gov, N. S.; Ideses, Y.; Bernheim-Groswasser, A. Eur. Biophys. J. 2008, 37, 447–454. (26) Grason, G. M.; Bruinsma, R. F. Phys. ReV. Lett. 2007, 99, 098101. (27) Gov, N. S. Phys. ReV. E 2008, 78, 011916.

10.1021/la803103k CCC: $40.75  2009 American Chemical Society Published on Web 12/30/2008

Polarity Sorting of Actin Bundles Formed with Polycation

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Experimental Procedures Materials. G-Actin was purified from scallops by the method of Spudich et al.28 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. Phalloidin binds to G-Actin stoichiometrically and stabilizes F-Actin against depolymerization at a low concentration of G-Actin. Myosin was obtained from scallops by the method of Barany et al.29 Heavy meromyosin (HMM) was obtained by the method of Craig et al.30 Poly-L-lysine (later denoted as p-Lys) (Sigma) of various degrees of polymerization N was used as purchased. p-Lys was 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 prepared by mixing F-Actin solution (1 µL) with p-Lys and KCl solution (9 µL) to yield a 10 µL reaction mixture as in our previous paper.24 Actin concentration CA, expressed in terms of G-Actin concentration, was kept constant at 2.3 × 10-7 M; p-Lys concentration CP, expressed in terms of Lys monomer unit, and KCl salt concentration CS were varied as described in the figure captions. Samples were incubated at room temperature for 60 min except for the time evolution experiment. Observation of the Time Profile of the Thickness Growth. We observed the time profile of the thickness growth using a transmission electron microscope (TEM). TEM observation was performed using a H-7650 (Hitachi, Tokyo, Japan) at 80 kV acceleration voltage. A 10 µL amount of sample was dropped on carbon-coated grids (Nisshin EM Co., Tokyo), which were rendered hydrophilically by glow discharge in a reduced pressure. After 3 min on the grids, 2% (w/v) uranyl acetate was added momentarily to the sample, and after 2 min on the grids, the grids were air dried. The Actin bundle thickness D of the Actin bundle was an average over about 20 samples. Polarity Measurement. We estimated the polarity of the Actin bundle from the arrowhead-like pattern of Actin filaments decorated with HMM.23,30 We relaxed bundles to individual filaments properly and decorated the filaments with HMM by the following procedures. The Actin bundle solution was centrifuged for 30 min at 65 000 rpm (Hitachi ultracentrifuge, himac CP70MX; Roter, R65A) to remove extra polycation in order to prevent its interaction with HMM and forming aggregates. A drop of solution containing the Actin bundle of about 10 µL was placed on carboncoated 200 mesh grids (Nisshin EM Co., Tokyo). The grids were coated with molten dental wax, which made the surface of the grid hydrophobic to prevent adsorption of free HMM to the grid.30 After waiting for 10-40 s, two drops of HMM buffer (5 mM phosphate (pH 7.0), 20 mM NaCl, 1 mM MgCl2, 0.1 mM EGTA, 2 mM NaN3) were placed on the grid in order to remove excess polymer-Actin mixture. Then one drop of HMM (0.1-0.5 mg/mL in HMM buffer) was placed on the grid. In this process, the Actin bundles were properly relaxed to individual filaments by decoration of HMM, and these filaments form an arrowhead-like structure with HMM along the polar direction. After waiting ∼10s the grids were washed by four drops of HMM buffer and 10 mM NaPi (pH 7.2). After washing, the grids were stained by four drops of 2% (w/v) uranyl acetate (pH 4.0). The samples were observed by TEM (H-7650) at a 80 kV acceleration voltage. The polarity P of the Actin bundle was defined by the following equation

P)

|n1 - n2| n1 + n2

where n1 and n2 are the numbers of Actin filaments pointing in the two opposite directions in an Actin bundle. The polarity, P, is the average value over n samples, where n ranges from 12 to 15. (28) Spudich, J. A.; Watt, S. J. Biol. Chem. 1971, 246, 4866–4871. (29) Barany, M.; Barany, K. Biochem. Z. 1966, 345, 37–56. (30) Craig, R.; Szent-Gyorgi, A. G.; Beese, L.; Flicker, P.; Vibert, P.; Cohen, C. J. Mol. Biol. 1980, 140, 35–55.

Figure 1. TEM images of Actin bundles formed with p-Lys of N ) 26 at CP ) 10-5 M, CS ) 0.01 M that was undecorated (a) and decorated (b) with HMM. In b the Actin bundle was properly relaxed to individual filaments and the arrowhead-like pattern was observed in these filaments by decoration of HMM. White arrows in b indicate the direction of arrowhead structures of decorated Actin filaments.

Figure 2. Thickness D and polarity P of the Actin bundles formed with p-Lys of N ) 26 in various CS at a constant CP ) 10-5M (a) and various CP at a constant CS ) 0.01 M (b). The Actin concentration was kept constant at 2.3 × 10-7 M. The error bars indicate the standard deviation.

Results and Discussion Figure 1a shows TEM images of the Actin bundle obtained by mixing F-Actins with p-Lys for 60 min. By decoration of HMM, the Actin bundle was properly relaxed to individual filaments and the arrowhead-like pattern of these filaments could be observed (Figure 1b). Figure 2 shows the bundle thickness D (left axis) and polarity P (right axis) of the Actin bundles formed with p-Lys (N ) 26) at various KCl concentration CS (Figure 2a) and p-Lys concentration CP (Figure 2b). As shown in Figure 2a, the D increases with CS but P barely changes with CS at a constant CP of 10-5 M. Salts effectively screen the electrostatic interaction and hinder the polycation-mediated attraction between Actins. According to the anisotropic nucleationgrowth mechanism, the weaker the attraction between F-Actins mediated by polycations, the larger the critical nuclei; therefore, the thicker the bundles formed.24 The increase in D with CS is in agreement with this explanation. The result shown in Figure 2a indicates that the strength of the electrostatic attraction between Actin and polycation has less effect on the polarity of the Actin bundle, and P and D are dominated by different factors, that is, the former does not depend on the strength of the electrostatic interaction but the latter does. In contrast, at a constant CS of 0.01 M P decreases with an increase in CP while D is insensitive to CP (Figure 2b). The latter phenomenon is in agreement with the anisotropic nucleation mechanism. The change of CP would influence the kinetics of nuclei formation of the Actin bundle since the kinetics is related to the collision frequency between F-Actin and polycation, which is a function of k, CP, and CA (where k is the reaction rate constant of nuclei formation).

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Figure 3. Time evolution of thickness D of the Actin bundle formed with p-Lys of N ) 1338 at various CS and CP. The Actin bundle formation was performed in (a) CP ) 10-6 M and CS ) 0.01 M; (b) CP ) 10-6 M and CS ) 0.1M; (c) CP ) 10-4 M and CS ) 0.01 M; (d) CP ) 10-4 M and CS ) 0.1 M. CA was kept constant at 2.3 × 10-7 M. The error bars indicate the standard deviation.

Since the D growth is finished in the nucleation stage, we observe the time profile of D growth to estimate the kinetics of nuclei formation. We found that the nucleation process of the Actin bundle formed with p-Lys of N ) 26 was finished within 5 min, which was our experimental resolution time. This prevents us from investigating the details of the nucleation kinetics.

Shikinaka et al.

Therefore, we used p-Lys of a larger molecular weight N ) 1338 and 76 instead of that of N ) 26 in this experiment for delaying the nucleation kinetics of the Actin bundle. Figure 3 shows the effect of CS and CP on the time profile of D growth of the Actin bundle. For a constant CP of 10-6 M, D growth progresses gradually up to 20 min regardless of increasing CS from 0.01 (Figure 3a) to 0.1 M (Figure 3b), while the final D size increases from 23.4 to 30.6 nm. In contrast, at a high CP of 10-4M the D growth is completed within 5 min, which is at least 4 times shorter than that at CP ) 10-6 M, while the time at which D growth is completed does not change with CS (Figure 3c and 3d). Figure 4 shows the nucleation time tN at which the D reaches plateau of Actin bundles at various CP and CS (Figure 4). As shown in these figures, tN decreases with an increase in CP (Figure 4a-c) but barely changes with CS (Figure 4d-f). These results indicate that the kinetics of nuclei formation changes with CP but not with CS. Thus, we conclude that the polarity of the Actin bundle is affected by the kinetics of nuclei formation from the results of Figures 2, 3, and 4. The characteristic length of p-Lys we employed here is much smaller than that of an F-Actin (several micrometers) because the gyration radius RF of p-Lys is less than several decades of nanometers according to polymer theory, RF ∝ aNν (where a is the segment length of p-Lys, N the degree of polymerization of p-Lys, V ) 0.5-10 depending on the rigidity of the polymer). Thus, the diffusion coefficient Ddiff, which is inversely proportional to the RF of molecules, of F-Actin is much smaller than that of p-Lys. This indicates that the kinetics of nuclei formation is limited by diffusion of the polycation. Therefore, assuming this diffusion-limited reaction by polycation the reaction rate constant k should decrease with an increase in the N of polycation. As shown in Figure 4d and 4f, tN for N ) 73 is about 2 times shorter than that for N ) 1338 at a constant CP of 10-6 M. This result confirms that the nucleation kinetics decreases with the increase in N at a constant CP. Furthermore, the tendency that the tN

Figure 4. Nucleation time tN of the Actin bundles formed with p-Lys at various CP, CS, and N. The Actin concentration was kept constant at 2.3 × 10-7 M. The interval of time between plots is 5 min, which was our experimental resolution time.

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process, corresponding to the nucleation process of the present work, might be due to the very quick kinetic process. Actually, formation of ParM bundle was finished within 200 s, which is much shorter than the shortest nucleation time in the present work (5 min). Similar phenomena have also been reported on Actin raft, which is a two-dimensional array of Actin filaments on positively charged lipid layers.33 In this raft the order of the parallel array increases with an increase in the fluidity of the lipid layer.33,34 This means that Actins prefer to form a unipolar array under appropriate conditions, that is, the F-Actin bundles in parallel are more stable than that in antiparallel. The kinetic effect on the polarity is consistent with the general principle with crystallization in which the quicker a crystal grows the poorer the crystallinity of the crystal is.35 Figure 5. Polarity P of Actin bundles formed with p-Lys of various degrees of polymerization N at the critical CP to form the bundle Ccp(55) and at a higher than Ccp (72); Ccp of N ) 5, 26, 186, and 1338 are 5.0 × 10-5, 5.0 × 10-6, 5.0 × 10-7, and 3.3 × 10-7 M, respectively, where Ccp is expressed in terms of lysine monomer unit. The CA and CS were kept constant at 2.3 × 10-7 and 0.01 M, respectively. n is the samples averaged. The lines are drawn to guide the eyes.

decreases with an increase in CP (Figure 4c) but barely changes with CS (Figure 4f) is the same for N ) 1338. As has been clarified, with an increase in N the critical CP to form bundle also decreases.24 Figure 5 shows the relationship between P and CP of the Actin bundle formed with p-Lys of various N just above the critical CP to form bundle. As shown in Figure 5, P increases with decreasing CP, the same as the result in Figure 2. Moreover, P also increases with increasing N at a constant CP (as in the case of N ) 5 and 26 at a CP of 10-4 M). These results lead to the conclusion that the polarity of the Actin bundle decreases with an increase in the kinetics of nuclei formation, which is proportional to Ddiff of polycation and CP. Previously, bundle formation of the Actin-like protein filaments ParM in the presence of crowding agent PVA (polyvinyl alcohol) was reported.31,32 These authors revealed, based on a dual color annealing experiment, that bundle formation is through the sideby-side random annealing process. This random annealing (31) Popp, D.; Gov, N. S.; Iwasa, M.; Mae´da, Y. Biopolymer 2008, 89, 711– 721. (32) Popp, D.; Yamamoto, A.; Iwasa, M.; Narita, A.; Maeda, K.; Mae´da, Y. Biochem. Biophys. Res. Commun. 2007, 353, 109–114.

Conclusions The polarity of the Actin bundle formed with p-Lys depends not on salt concentration but on polycation concentration. Furthermore, the polarity increases with increasing degree of polymerization of p-Lys at a constant polymer concentration. From these results it can be concluded that the kinetics of nuclei formation determines the polarity of the Actin bundle. It is considered that the polarity of the Actin bundle is essential for cell activity and that the cellular process would depend on how the unipolar structure of the Actin bundle is formed. Therefore, understanding the factors determining the polarity of the Actin bundle is important for the cellular processes. Many researchers have postulated that the specificity of Actin linker proteins decides the polarity of the Actin bundle. From our studies, the kinetics of Actin bundle formation also influences its polarity. Thus, it remains to be investigated experimentally whether the polarity sorting based on the kinetics of bundle formation actually occurs in living systems. Acknowledgment. We gratefully acknowledge the Ministry of Education, Science, Sports, and Culture, Japan (Grant-in-Aid of Specially Promoted Scientific Research) for financial support of this research. We are grateful to Dr. Hidemitsu Furukawa for his advice and help. LA803103K (33) Taylor, K. A.; Taylor, D. W. J. Struct. Biol. 1992, 108, 140–147. (34) Ward, R. J.; Menetret, J.-F.; Pattus, F.; Leonard, K. J. Electron Microsc. Tech. 1990, 14, 335–341. (35) Stokes, D. L.; DeRosier, D. J. Biophys. J. 1991, 59, 456–465.