Langmuir 2007, 23, 6257-6262
6257
Actin Network Formation by Unidirectional Polycation Diffusion Hyuck Joon Kwon,† Akira Kakugo,† Takehiro Ura,† Takaharu Okajima,‡ Yoshimi Tanaka,§ Hidemitsu Furukawa,† Yoshihito Osada,† and Jian Ping Gong*,†,| Department of Biological Science, Graduate School of Science, Hokkaido UniVersity, Sapporo 060-0810, Japan, Nanotechnology Research Center, Research Institute for Electronic Science, Hokkaido UniVersity, Sapporo 001-0021, Japan, CreatiVe Research InitiatiVe, Sapporo 001-0021, Japan, and SORST, JST, Sapporo 060-0810, Japan ReceiVed NoVember 23, 2006. In Final Form: February 26, 2007 We show that F-actins form three-dimensional giant network under uni-directional diffusion of polycations, at a dilute actin concentration (0.01 mg/mL) that only bundles are formed by homogeneous mixing with polycations. The mesh size of the actin network depends on polycation concentration and ionic strength, while bundle thickness of network depends only on ionic strength, which indicates that actin network is formed through nucleation-growth mechanism. The mesh size and the bundle thickness are determined by nucleus concentration and nucleus size, respectively. The atomic force microscopy measurement correlates the elasticity of the actin network, E, with the mesh size, ξ, as E ∼ ξ-1, while the bundle thickness, D dependence of E cannot be described by a simple scaling relation. E ∼ D6.5 when D is small and E ∼ D0.1 when D is large. Our study on the self-assembly of actin network under asymmetric polycation condition would provide the crucial insight into the organization of biopolymers in polarized condition of cell.
Introduction Biopolymers such as DNA, filamentous actins (F-actin), and microtubules behave as polyelectrolytes with negative charges in physiological condition.1,2 It has been known that a considerable number of cationic proteins are involved in the organization of these negatively charged biopolymers such as gene packaging and the cytoskeleton organization.3-9 This fact implies that electrostatic interaction plays a significant role in organization of biopolymers in vivo. In vitro studies have also found that these negatively charged biopolymers can be assembled into bundles, toroids, and networks by electrostatic interaction with multivalent cations, polycations, and cationic lipids.1-2,10-18 However, a physical principle for * To whom correspondence should be addressed. E-mail: gong@ sci.hokudai.ac.jp. Phone and Fax: 81(11)706-2774. † Graduate School of Science, Hokkaido University. ‡ Research Institute for Electronic Science, Hokkaido University. § Creative Research Initiative. | SORST, JST. (1) Tang, J. X.; Janmey, P. A. J. Biol. Chem. 1996, 271, 8556-8563. (2) Tang, J. X.; Ito, T.; Tao, T.; Traub, P.; Janmey, P. A. Biochemistry 1997, 36, 12600-12607. (3) Huttelmaier, S.; Harbeck, B.; Steffens, N. O.; Meberschmidt, T.; Illenberger, S.; Jockusch, B. M. FEBS Lett. 1999, 451, 68-74. (4) Harbeck, B.; Huttelmaier, S.; Schluter, K.; Jockusch, B. M.; Illenberger, S. J. Biol. Chem. 2000, 275, 30817-30825. (5) Tang, J. X.; Szymanski, P.; Janmey, P. A.; Tao, T. Eur. J. Biochem. 1997, 247, 432-440. (6) Amann, K. J.; Renley, B. A.; Ervasti, J. M. J. Biol. Chem. 1998, 273, 28419-28423. (7) Goldmann, W. H.; Guttenberg, Z.; Tang, J. X.; Kroy, K.; Isenberg, G.; Ezzell, R. M. Eur. J. Biochem. 1998, 254, 413-419. (8) Earnshaw, W. C.; King, J.; Harrison, S. C.; Eiserling, F. A. Cell 1978, 14, 559-568. (9) Rao, V. B.; Black, L. W. J. Mol. Biol. 1985, 185, 565-578. (10) Needleman, D. J.; Ojeda-Lopez, M. A.; Raviv, Uri.; Miller, H. P.; Wilson, L.; Safinya, C. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 101, 16099-16103. (11) Raviv, U.; Needleman, D. J.; Li, Y.; Miller, H. P.; Wilson, L.; Safinya, C. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11167-11172. (12) Bloomfield, V. A. Biopolymers 1991, 31, 1471-1481. (13) Yoshikawa, K.; Yoshikawa, K.; Kanbe, T. Langmuir 1999, 15, 40854088. (14) Conwell, C. C.; Vilfan, I. D.; Hud. N. V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9296-9301. (15) Conwell, C. C.; Hud. N. V. Biochemistry 2004, 43, 5380-5387.
how self-organization of biopolymers is determined has been poorly understood, despite a considerable number of theoretical explanations. Moreover, most of the research has been concentrated in the formation of DNA toroids.12-15 We have investigated systematically actin bundle formation by interaction with various polycations and showed that F-actins form bundles above a critical polycation concentration and growth in bundle thickness D is independent of that in bundle length L.19-21 Essential experimental results in systematic study on F-actin bundle formation are summarized as follows:21 (1) The growth in bundle thickness D is completed at the initial stage of bundle formation and the bundle length L dramatically increases later on, after the D growth. (2) D is weakly dependent on actin concentration (CA), while L increases with increase in CA. (3) D decreases remarkably with an increase in polycationmediated attraction between F-actins that is brought about by an increase in the polycation polymerization degree or a decrease in salt concentration. Results (1) and (2) indicate that the self-assembly consists of two stages, and D and L are determined by different factors, respectively; and result (3) indicates that D is not determined by general thermodynamics. From these results, we concluded that the first stage of the actin bundle formation is the nucleation process and the second stage is the growth process, and we proposed the anisotropic nucleation-growth model in which D is determined in the nucleation process by the critical nucleus size D0, while L is determined by free F-actins available to growth relative to the concentration of stable nuclei.21 (16) Wong, G. C. L.; Lin, A.; Tang, J. X.; Li, Y.; Janmey, P. A.; Safinya, C. R. Phys. ReV. Lett. 2003, 91, 018103 (1-4). (17) Wong, G. C. L.; Tang, J. X.; Lin, A.; Li, Y.; Janmey, P. A.; Safinya, C. R. Science 2000, 288, 2035-2039. (18) Sowa, G. Z.; Cannell, D. S.; Liu, A. J.; Reisler, E. J. Phys. Chem. B 2006, 110, 22279-22284. (19) Kakugo, A.; Shikinaka, K.; Matsumoto, K.; Gong, J. P.; Osada. Y. Bioconjug. Chem. 2003, 14, 1185-1190. (20) Kwon, H. J.; Kakugo, A.; Shikinaka, K.; Furukawa, K.; Osada, Y.; Gong. J. P. Biomacromolecules 2005, 6, 3005-3009. (21) Kwon, H. J.; Tanaka, Y.; Kakugo, A.; Shikinaka, K.; Furukawa, K.; Osada, Y.; Gong. J. P. Biochemistry 2006, 45, 10313-10318.
10.1021/la063416k CCC: $37.00 © 2007 American Chemical Society Published on Web 04/27/2007
6258 Langmuir, Vol. 23, No. 11, 2007 Scheme 1. Chemical Structure of PDMAPAA-Q
Actin architectures such as bundle and network are organized, determining mechanical property of a cell, changing cells shape, and guiding cell motility in vivo.22 Intrinsic polarity of cells enables signal molecules such as Rho family GTPase and components such as WASP family and Arp2/3 complex involved in the organization of actin architectures to exhibit a strong localization within a cell.23-25 This cellular asymmetric condition induces the asymmetric organization of actin architectures. Thus, it is important to investigate self-organization of actin architectures under asymmetric or localized activation conditions. In this paper we describe the self-assembly of F-actins under asymmetric conditions by unidirectional diffusion of polycations to F-actin solution. We show that F-actins are effectively assembled into a globally linked network under unidirectional diffusion of polycation by using a microchamber or polymer gel, even at a low actin concentration that only small actin bundles are formed under homogeneous mixing of F-actins with polycations. The mesh size (ξ) of actin network depends strongly on both polycation concentration (CP) and salt concentration (CS), while the bundle thickness (D) of actin network depends strongly only on CS. Additionally, atomic force microscopy (AFM) reveals the correlation between network structure and the elastic modulus of a three-dimensional (3D) actin network. This study shows that F-actins are assembled into an actin network by a nucleation-growth mechanism, where the selforganization of actin architectures is strongly influenced kinetically by spatiotemporal control of nucleation, and the actin network produces stable mechanical property even at a low actin concentration, which is influenced by the structural property of the network. Experimental Section Materials. G-actin was purified from scallops by the method of Spudich and Watt.26 Fluorescence-labeled 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, 100 mM KCl, and 2 mM MgCl2) for 24 h at 4 °C. Phalloidin binds to G-actin stoichiometrically and stabilizes F-actin against depolymerization at a decreased critical concentration of actin. Poly-N-[3-(dimethylamino)propyl]acrylamide methyl chloride quaternary (PDMAPAA-Q), which has the chemical structure as shown in Scheme 1, was prepared by radical polymerization of a 1.0 M aqueous solution of DMAPAA-Q monomer (Tokyo Kasei Co., Ltd) in the presence of 0.2 mol % potassium persulfate (Tokyo Kasei Co., Ltd). PDMAPAA-Q has Mw ∼ 184000 (valence: ca. 873) and the radius of gyration ca. 40 nm, which were measured by static light scattering. PDMAPAA-Q is diluted by HEPES (pH 7.2) buffer and used for experiments. (22) Howard, J. Mechanics of motor proteins and the cytoskeleton; Sinauer Associates. Inc.: Sunderland, Massachusetts, 2001; pp 110-116. (23) Chung, C. Y.; Funamoto, S.; Firtel, R., Trends Biochem. Sci. 2001, 26, 557-566. (24) Kurokawa, K.; Nakamura, T.; Aoki, K.; Matsuda, M. Biochem. Soc. Trans. 2005, 33, 631-634. (25) Svitkina, T.; Borisy, G. J. Cell. Biol. 1999, 145, 1009-1026. (26) Spudich, J. A.; Watt, S. J. Biol. Chem. 1971, 246, 4866-4871.
Kwon et al. Polyacrylamide (PAAm) gel was prepared by radical polymerization of a 2.0 M aqueous solution of acrylamide (AAm) (Tokyo Kasei Co., Ltd) in the presence of 0.2 mol % 2-oxoglutaric acid and 0.25 mol % N,N′-methylenebisacrylamide (MBAA) (Tokyo Kasei Co., Ltd), which was recrystallized twice. The gelation was carried out by UV light for 24 h under a nitrogen atmosphere. Actin Network Formation in a Microchamber. The microchamber (diameter:
