Selective Spatial Localization of Actomyosin Motor Function by

Jul 13, 2006 - Parallel computation with molecular-motor-propelled agents in nanofabricated networks. Dan V. Nicolau , Mercy ... Tracking Actomyosin a...
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Langmuir 2006, 22, 7302-7312

Selective Spatial Localization of Actomyosin Motor Function by Chemical Surface Patterning Mark Sundberg,† Martina Balaz,† Richard Bunk,‡ Jenny P. Rosengren-Holmberg,† Lars Montelius,*,‡ Ian A. Nicholls,† Pa¨r Omling,‡ Sven Tågerud,† and Alf Månsson*,† Department of Chemistry and Biomedical Sciences, UniVersity of Kalmar, SE-391 82 Kalmar, Sweden, and DiVision of Solid State Physics and The Nanometer Consortium, UniVersity of Lund, Box 118, SE-221 00 Lund, Sweden ReceiVed February 7, 2006. In Final Form: May 25, 2006 We have previously described the efficient guidance and unidirectional sliding of actin filaments along nanosized tracks with adsorbed heavy meromyosin (HMM; myosin II motor fragment). In those experiments, the tracks were functionalized with trimethylchlorosilane (TMCS) by chemical vapor deposition (CVD) and surrounded by hydrophilic areas. Here we first show, using in vitro motility assays on nonpatterned and micropatterned surfaces, that the quality of HMM function on CVD-TMCS is equivalent to that on standard nitrocellulose substrates. We further examine the influences of physical properties of different surfaces (glass, SiO2, and TMCS) and chemical properties of the buffer solution on motility. With the presence of methylcellulose in the assay solution, there was HMM-induced actin filament sliding on both glass/SiO2 and on TMCS, but the velocity was higher on TMCS. This difference in velocity increased with decreasing contact angles of the glass and SiO2 surfaces in the range of 20-67° (advancing contact angles for water droplets). The corresponding contact angle of CVD-TMCS was 81°. In the absence of methylcellulose, there was high-quality motility on TMCS but no motility on glass/SiO2. This observation was independent of the contact angle of the glass/SiO2 surfaces and of HMM incubation concentrations (30-150 µg mL-1) and ionic strengths of the assay solution (20-50 mM). Complete motility selectivity between TMCS and SiO2 was observed for both nonpatterned and for micro- and nanopatterned surfaces. Spectrophotometric analysis of HMM depletion during incubation, K/EDTA ATPase measurements, and total internal reflection fluorescence spectroscopy of HMM binding showed only minor differences in HMM surface densities between TMCS and SiO2/glass. Thus, the motility contrast between the two surface chemistries seems to be attributable to different modes of HMM binding with the hindrance of actin binding on SiO2/glass.

Introduction Force generation and the shortening of muscle is produced by cyclic interactions between myosin II motor proteins and actin filaments driven by the hydrolysis of ATP.1 The myosin motors in muscle are assembled into thick filaments that slide relative to the actin filaments during contraction.2,3 Because of this arrangement and the further assembly of the actin and myosin filaments into a hexagonal lattice, force production occurs with the myosin motors and actin filaments in a highly ordered arrangement. The in vitro motility assay4-8 that was developed in the 1980s9,10 is a valuable experimental system for the study of actomyosin function. In the most frequently used version, * Corresponding authors. (A.M.) E-mail: [email protected]. Tel: +46480-446243. Fax: +46-480-446262. (L.M.) E-mail: [email protected]. † University of Kalmar. ‡ University of Lund. (1) Geeves, M. A.; Fedorov, R.; Manstein, D. J. Cell Mol. Life Sci. 2005, 62, 1462-1477. (2) Huxley, H.; Hanson, J. Nature 1954, 173, 973-976. (3) Huxley, A. F.; Niedergerke, R. Nature 1954, 173, 971-973. (4) Tsiavaliaris, G.; Fujita-Becker, S.; Manstein, D. J. Nature 2004, 427, 558561. (5) Wells, A. L.; Lin, A. W.; Chen, L. Q.; Safer, D.; Cain, S. M.; Hasson, T.; Carragher, B. O.; Milligan, R. A.; Sweeney, H. L. Nature 1999, 401, 505-508. (6) Homsher, E.; Wang, F.; Sellers, J. R. Am. J. Physiol. 1992, 262, C714C723. (7) Harada, Y.; Sakurada, K.; Aoki, T.; Thomas, D. D.; Yanagida, T. J. Mol. Biol. 1990, 216, 49-68. (8) Uyeda, T. Q.; Kron, S. J.; Spudich, J. A. J. Mol. Biol. 1990, 214, 699-710. (9) Kron, S. J.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A 1986, 83, 62726276. (10) Kron, S. J.; Toyoshima, Y. Y.; Uyeda, T. Q.; Spudich, J. A. Methods Enzymol. 1991, 196, 399-416.

fluorescently labeled actin filaments are observed as they slide on a surface coated with randomly oriented myosin motors. In most studies (e.g., this work), the proteolytic heavy meromyosin (HMM) motor fragment is used rather than the entire myosin molecule. The in vitro assay, in contrast to in vivo studies, enables detailed control of the chemical environment and the use of protein engineering4 and single-molecule force measurements.11-13 However, a key limitation of the assay is the disordered adsorption of the motor proteins on the surface with potentially important disturbances of the mechanisms of motor function.14 To study certain cooperative aspects of the function in vitro, the myosin motors should ideally be positioned with great precision in a 3D arrangement similar to that in the muscle sarcomere. Well-defined positioning is also important if the aim is to exploit the motor proteins as nanoactuators or cargo transporters in lab-on-a-chip devices and similar applications.15-24 (11) Molloy, J. E.; Burns, J. E.; Kendrick-Jones, J.; Tregear, R. T.; White, D. C. Nature 1995, 378, 209-212. (12) Finer, J. T.; Simmons, R. M.; Spudich, J. A. Nature 1994, 368, 113-119. (13) Kishino, A.; Yanagida, T. Nature 1988, 334, 74-76. (14) Tanaka, H.; Ishijima, A.; Honda, M.; Saito, K.; Yanagida, T. Biophys. J. 1998, 75, 1886-1894. (15) Bunk, R.; Klinth, J.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tagerud, S.; Mansson, A. Biochem. Biophys. Res. Commun. 2003, 301, 783-788. (16) Clemmens, J.; Hess, H.; Lipscomb, R.; Hanein, Y.; Bohringer, K. F.; Matzke, C. M.; Bachand, G. D.; Bunker, B. C.; Vogel, V. Langmuir 2003, 19, 10967-10974. (17) Månsson, A.; Sundberg, M.; Bunk, R.; Balaz, M.; Nicholls, I. A.; Omling, P.; Tegenfeldt, J. O.; Tågerud, S.; Montelius, L. IEEE Trans. AdV. Packag. 2005, 28, 547-555. (18) Bunk, R.; Klinth, J.; Rosengren, J.; Nicholls, I.; Tagerud, S.; Omling, P.; Mansson, A.; Montelius, L. Microelectron. Eng. 2003, 67-68, 899-904. (19) Bohm, K. J.; Stracke, R.; Muhlig, P.; Unger, E. Nanotechnology 2001, 12, 238-244. (20) Dennis, J. R.; Howard, J.; Vogel, V. Nanotechnology 1999, 10, 232-236.

10.1021/la060365i CCC: $33.50 © 2006 American Chemical Society Published on Web 07/13/2006

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One approach to achieving precise positioning of motors on a surface is to use top-down nanofabrication (e.g., electron beam lithography) and selective surface functionalization to guide the localized assembly of the motors. In this process, it is important to have access to a substrate to which the motors adsorb with minimal disturbance of their physiological function (i.e., their actin-propelling and ATP-hydrolyzing capabilities). For lab-ona-chip applications and functional studies, it is important that this substrate can be nanostructured, leaving patches of active areas surrounded by inactive areas with no motor function. In previous work,25 glass surfaces derivatized with trimethylchlorosilane (TMCS) were generally found to be better than the standard substrate nitrocellulose10 in supporting high-quality motility on nonpatterrned surfaces. We also showed that TMCS was superior to several other silanes in this respect,25 and surfaces derivatized with TMCS are also superior to polymer resists that were previously used to create nanostructured surfaces15 for actomyosin motility. Furthermore, we have observed in a number of studies15,25,26 that there is poor motility or no motility on hydrophilic surfaces with excess negative charge27,28 (e.g., glass, SiO2, and O2-plasma-treated PMMA). Without performing any detailed analysis of the basis for the selectivity in motility between TMCS and the hydrophilic surfaces, we recently26 utilized our observations to achieve spatial control on the nanometer scale of HMM-induced actin filament sliding. In earlier work, we achieved almost complete guidance and unidirectional actin filament sliding along TMCS-functionalized nanosized tracks that were surrounded by hydrophilic (O2-plasma-treated) PMMA. These nanostructured surfaces were created by electron beam lithography (EBL) and chemical vapor deposition of TMCS (CVD-TMCS). The silanziation using vapor-phase techniques, rather than solution-phase processes, as studied previously by Sundberg et al.25 was deemed necessary for use in this case.26 Future goals will be to produce nanostructured surfaces with CVD-TMCS and hydrophilic areas optimized for biophysical studies of actomyosin function and for lab-on-a-chip applications. For use in biophysical studies, it is first important to extend our previous results26 to confirm that CVD-TMCS (like liquid-phase deposited TMCS) supports actomyosin motility that is similar in quality to that on standard nitrocellulose substrates (e.g., refs 6, 10, and 29). Both for the interpretation of functional studies and for the optimization of lab-on-a-chip applications, it is also important to establish the conditions that allow motility selectivity between hydrophobic TMCS and hydrophilic surfaces. Finally, of particular importance for functional studies, it is important to understand the mechanisms that underlie the selectivity. In the present work, we investigate the above issues using glass and SiO2 as model hydrophilic surfaces with negative charge. Our studies provide firm evidence that actomyosin motility is of similar quality on vapor-deposited TMCS as on nitrocel(21) Hess, H.; Clemmens, J.; Qin, D.; Howard, J.; Vogel, V. Nano Lett. 2001, 1, 235-239. (22) Hiratsuka, Y.; Tada, T.; Oiwa, K.; Kanayama, T.; Uyeda, T. Q. Biophys. J. 2001, 81, 1555-1561. (23) Jia, L. L.; Moorjani, S. G.; Jackson, T. N.; Hancock, W. O. Biomed. MicrodeVices 2004, 6, 67-74. (24) Suzuki, H.; Yamada, A.; Oiwa, K.; Nakayama, H.; Mashiko, S. Biophys. J. 1997, 72, 1997-2001. (25) Sundberg, M.; Rosengren, J. P.; Bunk, R.; Lindahl, J.; Nicholls, I. A.; Tagerud, S.; Omling, P.; Montelius, L.; Mansson, A. Anal. Biochem 2003, 323, 127-138. (26) Bunk, R.; Sundberg, M.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Månsson, A.; Montelius, L. Nanotechnology 2005, 16, 710-717. (27) Bismarck, A.; Boccaccini, A. R.; Egia-Ajuriagojeaskoa, E.; Hulsenberg, D.; Leutbecher, T. J. Mater. Sci. 2004, 39, 401-412. (28) Chai, J. N.; Lu, F. Z.; Li, B. M.; Kwok, D. Y. Langmuir 2004, 20, 1091910927. (29) Klinth, J.; Arner, A.; Mansson, A. J. Muscle Res. Cell Motil. 2003, 24, 15-32.

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lulose. We also examined the conditions (HMM incubation concentrations, solution composition, contact angle of surfaces, etc.) for motility selectivity between vapor-deposited TMCS and SiO2/glass. Selectivity was achieved for a range of HMM incubation concentrations and ionic strengths and for a range of contact angles of the SiO2/glass areas provided that methylcellulose was not present in the assay solutions. Importantly, similar conditions allowed the selective localization of function to TMCS areas on micro- and nanopatterned surfaces. Our results suggest that the mechanisms underlying the selectivity involves differences in the mode of HMM adsorption between TMCS and SiO2/ glass with only small differences in the HMM density. The results are discussed in relation to previous models for HMM binding to nitrocellulose surfaces.30 The implications for lab-on-a-chip applications and nanotechnology-based assay systems for functional studies of actomyosin are also discussed. Brief accounts of some aspects of the present work were given earlier.31,32 Experimental Methods Surface Preparation and Characterization by Contact Angle Measurements. Cover glasses (Schott D263M; Schott AG, Mainz, Germany) were immersed in 70% ethanol for >12 h and were blown dry with nitrogen. Nitrocellulose surfaces were prepared as described previously.25 Silicon dioxide surfaces were prepared on Si〈100〉 wafers by oxidation to a SiO2 thickness of 100-800 nm. Silanization of nonpatterned Si/SiO2 wafer surfaces and cover glasses was then achieved by the deposition of trimethylchlorosilane (TMCS) from solution25 or by chemical vapor deposition (CVD). In the latter case, the surface was placed for 7-60 min in a reaction chamber (and in turn placed in a glovebox) with a TMCS-saturated atmosphere. The water contact angle and HMM-induced actin motility were independent of silanization time as tested in separate experiments. Glass and SiO2 surfaces functionalized with TMCS will, in the following text, be referred to as TMCS surfaces. Surfaces with TMCS micropatterns were prepared by UV lithography on oxidized Si wafers spin coated with resist S1813 (Shipley Company, Marlborough, MA), followed by development and chemical vapor deposition of TMCS. (For details, see ref 26.) The PMMA was removed using an organic cleaning cycle involving chloroform or hexane, acetone, and finally a rinse in 2-propanol or methanol. Nonpatterned TMCS and SiO2 surfaces were usually cleaned in a similar way. However, in some experiments nonsilanized SiO2 wafers were subjected to piranha cleaning (concd H2SO4 and 30% H2O2 in a 7:3 ratio, 80 °C, 5 min) to produce a more effective reduction in the contact angle. Caution: piranha solution is a highly corrosiVe acidic solution that can react Violently with organic materials. Do not store in a closed container, and use appropriate safety precautions. All silicon wafers and glass surfaces (whether silanized or not) were placed in deionized water for at least 30 min before use in the motility assay or for contact angle measurements. TMCS nanopatterns (details in ref 26) were created on Si/SiO2 wafers. Rectangular TMCS loading zones (50 × 100 µm2) were produced for the initial binding of actin filaments. These TMCS loading zones were continuous with nanometer-wide (150-500 nm) TMCS tracks. The nanopatterns were created essentially as described above for the TMCS/SiO2 micropatterns, but electron beam lithography (EBL) was used for the patterning process rather than UV lithography. Advancing and receding contact angles were determined for nonpatterned glass, SiO2, and TMCS surfaces as described in ref 25 and in greater detail in the Supporting Information. (30) Toyoshima, Y. Y. AdV. Exp. Med. Biol. 1993, 332, 259-265. (31) Månsson, A.; Sundberg, M.; Bunk, R.; Balaz, M.; Rosengren, J. P.; Lindahl, J.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Montelius, L. Biophys. J. 2004, 86, 58a. (32) Månsson, A.; Sundberg, M.; Bunk, R.; Balaz, M.; Nicholls, I. A.; Tagerud, S.; Omling, P.; Montelius, L. J Muscle Res. Cell Motil. 2004, 25, 251.

7304 Langmuir, Vol. 22, No. 17, 2006 Protein Preparations and In Vitro Motility Assays. Actin and myosin were purified from rabbit skeletal muscle,33,34 with the myosin obtained exclusively from white leg muscles.35 For some of the experiments, actin was also obtained from bovine cardiac muscle.36 Flow cells with patterned or nonpatterned surfaces were prepared, and motility assays were performed essentially as described previously.15,25,35 A “wash solution” (25 mM imidazole hydrochloride at pH 7.4, 4 mM MgCl2, 1 mM EGTA, 25 mM KCl) was used to dilute the protein stock solutions and for rinsing between some incubation steps. The flow cell was incubated with two volumes each of the following solutions: HMM (30-200 µg mL-1, 2 min), bovine serum albumin (BSA; 1 mg mL-1, 30 s), blocking actin (1-5 µM, 1 min; to block rigor heads), assay solution a40 (see below; 30 s), wash solution, tetramethylrhodamine-phalloidin (RhPh)-labeled actin (2-6 nM, 30 s), wash solution, and finally an assay solution (see below) to initiate filament movement. The blocking actin was sheared by repeated passage through a 27 gauge needle before the experiments. Except for few experiments on micro- and nanopatterned surfaces that were carried out at room temperature (as specified below), the temperature varied between 28.3 and 31.2 °C. In each given experiment, the temperature was constant to (0.7 °C. When incubating the patterned surfaces, 20% of the blocking actin was labeled with Alexa488-phalloidin (APh; emitting green light) to facilitate pattern visualization without later preventing the observation of RhPh actin (orange-red emission) at low concentration. APh-labeled blocking actin will be referred to as APh actin in the following text. Actomyosin function has been shown to be effectively identical using either RhPh or APh labeling.36 Assay solutions (1 mM MgATP, pH 7.4) with and without methylcellulose were used. The assay solution with methylcellulose (aMC130) had an ionic strength of 130 mM whereas those without methylcellulose had ionic strengths from 20 to 50 mM (denoted by a20, a30, a40, and a50). In vitro motility assay experiments were included in the data analysis only if the fraction of motile filaments on a standard nitrocellulose surface (tested in each experiment) exceeded 0.60. In some experiments where the long-term observation (>15-30 min) of actomyosin function was required, creatine phoshphate (2.5 mM) and creatine kinase (56 units/mL) were added to the assay solutions. The fraction of motile filaments and the sliding velocities were analyzed by automatic and semiautomatic computer algorithms.29,37 Filament lengths were measured for studies of the length dependence of sliding velocities and for the estimation of the quantum yield of APh-labeled actin filaments. First, background subtraction was performed, yielding the integrated light intensity of a filament in a given frame.38 From these data, the true filament length (lt) of the sliding filaments was calculated using a standard curve. The coefficient of variation in the estimate of lt for short filaments (0.10.5 µm) was about 20%. More details of protein preparations and in vitro motility assays (e.g., assay solutions, recording of data, measurement of filament lengths, and analysis) are given in Supporting Information. HMM Surface Density. A. K/EDTA-ATPase ActiVity. The density of catalytically active HMM molecules on flow cell surfaces was determined by measuring the K/EDTA-ATPase activity8 essentially as described previously39 using a colorimetric method40 to measure inorganic phosphate. In a majority of the experiments, we obtained only the relative values for the HMM density on TMCS and cover (33) Pardee, J. D.; Spudich, J. A. Methods Cell Biol. 1982, 24, 271-289. (34) Sata, M.; Sugiura, S.; Yamashita, H.; Momomura, S.; Serizawa, T. Circ. Res. 1993, 73, 696-704. (35) Klinth, J.; Arner, A.; Mansson, A. J. Muscle Res. Cell Motil. 2003, 24, 15-32. (36) Balaz, M.; Mansson, A. Anal. Biochem 2005, 338, 224-236. (37) Mansson, A.; Tagerud, S. Anal. Biochem 2003, 314, 281-293. (38) Månsson, A.; Sundberg, M.; Balaz, M.; Bunk, R.; Nicholls, I. A.; Omling, P.; Tagerud, S.; Montelius, L. Biochem. Biophys. Res. Commun. 2004, 314, 529534. (39) Toyoshima, Y. Y.; Kron, S. J.; Spudich, J. A. Proc. Natl. Acad. Sci. U.S.A 1990, 87, 7130-7134. (40) Kodama, T.; Fukui, K.; Kometani, K. J. Biochem. (Tokyo) 1986, 99, 1465-1472.

Sundberg et al. glass surfaces. In such experiments, the data were normalized to the HMM density on glass at saturation because data at saturating concentrations were not obtained for TMCS in all experiments. Two separate experiments (two different HMM preparations and two different batches of surface preparations) were performed to determine the absolute HMM surface densities at saturation (HMM incubation concentration 120 µg/mL). In these experiments, a 3 mM initial ATP concentration was used, and the K/EDTA-ATPase activity of HMM in flow cells was compared to the K/EDTA-ATPase activity of HMM in solution. The relationship between the amount formed Pi and the HMM concentration in solution (standard curve) was highly linear (r2 > 0.97) in both experiments. More details describing the K/EDTA-ATPase measurements are given in Supporting Information. B. HMM Depletion of the Incubation Solution. The surface adsorption of HMM on cover glasses and liquid-deposited TMCS was estimated by fluorescence spectroscopy from the HMM depletion of the incubation solution. Flow cells with large surface areas (∼10 cm2; height ∼100 µm) were incubated with HMM (130 µg/mL) for 2 min. After incubation, the flow cell was rinsed with wash solution, and the entire efflux volume was collected in a quartz cuvette, resulting in about a 10-fold dilution of the original incubation solution. This procedure achieved close to 100% recovery of the flow cell contents. This was indicated by one separate experiment that was observed to give 102% recovery based on the fluorescence of nonadsorbing Na-fluorescein included in the incubation solution. The incubation and efflux volumes were calculated from the weights (with and without solution) of the flow cell and cuvette, respectively. The amount of HMM adsorbed was calculated from the amount of HMM in the incubation solution before and after incubation. These amounts were measured by fluorescence spectroscopy (excitation at 280 nm and emission at 335 nm) using a standard curve (r2> 0.996) of known HMM concentrations. C. Fluorescence Spectroscopy Using a TIRF Accessory. The surface adsorption of HMM and subsequent binding of actin were investigated using fluorescence spectroscopy in a spectrofluorometer (HORIBA Jobin Yvon Inc., NJ; Total internal reflection fluorescence (TIRF) accessory from BioElectroSpec, PA). Measurements were performed on cleaned silica glass (fused silica; SiO2) and on silica glass silanized with TMCS using liquid deposition. Cleaning of the surfaces (the TIRF slides) was achieved using piranha solution (see above), followed by thorough rinsing in deionized water. This step was a prerequisite for effective subsequent silanization of the silica glass surfaces when carried out as previously described.25 The fluorescence spectroscopy cells were incubated essentially as in the in vitro motility assay procedure with, in sequence, HMM (130 µg/mL), wash solution, BSA (1 mg/mL), and wash solution again. During the incubation with HMM, the emitted fluorescence intensity was measured at 335 nm (excitation 280 nm). Thereafter, the cells were incubated with 2 nM RhPh-labeled actin, wash solution, 1 µM actin (APh-actin; 4% APh labeled), wash solution, a40 assay solution, and finally, wash solution again. The processes starting with the infusion of APh actin were monitored by excitation at 495 nm and the recording of emission at 519 nm. The same integration time (0.5 s) and sampling rate (0.5 s-1) were used in all measurements. Differences in the quantum yield of APh-labeled actin on SiO2 and TMCS are relevant to the interpretation of the fluorescence spectroscopy-based actin binding data. The differences in quantum yield between SiO2 and TMCS were assessed from the fluorescence intensity per filament length on these surfaces. Such measurements, in two different experiments, gave a higher quantum yield on TMCS by 15 ( 5% (n ) 6 filaments) and 40 ( 9% (n ) 9). For TIRF studies (evanescent wave excitation), the incident angle of the excitation light was set to about 70°, corresponding to a penetration depth of evanescent wave excitation of about 60 nm for a wavelength of 280 nm.41 The measurement of fluorescence intensity in the presence of high bulk concentrations of HMM suggested that the contribution of scattered light to excitation in the evanescent wave region (within 60 nm from the surface) corresponded to (41) Axelrod, D. Traffic 2001, 2, 764-774.

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considerably less than 1% of that attributed to excitation by the evanescent wave. This conclusion is based on the assumption that excitation by scattered light occurs uniformly for the entire depth of the spectroscopy cell. In some experiments, the incident angle was reduced below the critical angle for total internal reflection, resulting in the transmission of the excitation light. Under these conditions, there was relatively uniform excitation within a large part of the spectroscopy cell depth as suggested by comparison between HMM emission in the bulk and on the surface. While immersed in the wash solution, the TIRF slides were removed from the fluorescence spectroscopy cell in one of the experiments and then mounted in a flow cell for a motility assay. The flow cell was incubated with RhPh actin (2 nM, 30 s), wash solution, and a40 assay solution, and actin sliding was recorded as described above. Analysis of Data and Statistics. According to a model,8 the sliding velocity (Vf) of actin filaments moving over a myosin-coated surface (with myosin head density F) depends on the actin filament length (l) according to the following relationship: Vf ) ηV0{1 - (1 - f )Fld}

(1)

Here, V0 is the sliding velocity on a TMCS surface, and η is an effectiveness parameter determining the effectiveness of a given myosin cross-bridge to propel the filament (Discussion section). The quantity f is the duty ratio (i.e., the fraction of the ATPase cycle time that the myosin head spends strongly attached to actin). Finally, d is the width of a band surrounding the filament where interactions with adsorbed myosin motors is possible. In the present study, f and d were assumed to be 0.05 and 30 nm, respectively.8 On TMCS, the myosin head density F was estimated from K/EDTA ATPase measurements.8 The model was fitted to the data using nonlinear regression (Marquardt algorithm) in GraphPad Prism 4 (GraphPad Software Inc., CA). In the fitting procedure, η was first calculated from the average sliding velocity for large l (where the velocity was constant), and F was then obtained by minimizing the least-squares difference between the data and eq 1. Experimental data are presented as the mean ( the standard error of the mean (SE). Statistical hypothesis testing was performed using the two-tailed Student’s t-test. The paired t-test was used when applicable.

Results Actomyosin Motility Contrast between SiO2 and VaporDeposited TMCS Surfaces. Two different assay solutions were used in initial tests of contrast in HMM-induced actin filament sliding between SiO2 and CVD-TMCS. One of the assay solutions (aMC130) included 0.6% methylcellulose and had an ionic strength (130 mM) close to the physiological. The other (a40) had an ionic strength of 40 mM and contained no methylcellulose. On vapor-deposited TMCS, the advancing and receding contact angles were 81 ( 1 and 66 ( 1°, respectively (n ) 4 surfaces). On SiO2, the contact angles were lower but exhibited considerable variability (see below). Pooled data for motility quality on micropatterned and nonpatterned surfaces with TMCS and/or SiO2 are given in Figure 1A for HMM incubation concentrations of 60-200 µg mL-1. It can be seen here that both the sliding velocity and the fraction of motile filaments was similar on TMCS and nitrocellulose in a40 as well as in aMC130 assay solutions. In none of the experiments was there any motility on SiO2 in a40 solution (Figure 1A). This was due to the detachment of the filaments from the surface immediately upon infusion of the assay solution. In aMC130 assay solution, the motility quality on SiO2 was variable, but the sliding velocity was generally lower than on TMCS as exemplified by the micropatterned surface in Figure 1B. Here, a filament sliding on SiO2 can be seen to accelerate immediately upon entering the TMCS circle and then continue sliding at more

than twice the original velocity. The contrast in contact angle between TMCS and SiO2 in this case is illustrated by the inset in Figure 1B. The variability in the quality of motility on SiO2 in aMC130 solution may be attributable to different factors. First, there was apparent variation in the chemistries of the SiO2 surfaces as reflected by the different contact angles. This effect is illustrated in Figure 1C for nonpatterned glass and SiO2 surfaces where the contact angle could be directly quantified. Varying contact angles were here the results of different cleaning methods and/or variable storage times of the surfaces (Discussion). The variability applied both to the advancing and receding contact angles, but on average, the receding angle on SiO2 was 22 ( 2° (n ) 5) lower than the advancing angle. It can be seen in Figure 1C that the sliding velocity on glass and SiO2 decreased with a decrease in both advancing/static and receding contact angles. A second factor that may have contributed to the variability in sliding velocity on SiO2 is a more marked time dependence than on TMCS. In one experiment (Figure 1D), the average velocity on SiO2 was 90% of the velocity on TMCS immediately (within a few minutes) after the infusion of assay solution. This value decreased to 70% after a 20 min observation period and then remained at 70% after more than 2 h and several exchanges of assay solution. Under these conditions, the sliding velocity on TMCS was reduced by only 10%. In contrast to the case for aMC130 solution, there was, as mentioned above, always complete motility contrast in a40 solution (no sliding filaments on SiO2; Figure 1A). This contrast was observed both when there were small and large differences in the contact angle between SiO2 and TMCS (e.g., for the SiO2 surface with the highest contact angle in Figure 1C). The motility contrast between the TMCS and SiO2 in a40 assay solution was seen both for micro- and nanopatterned surfaces as exemplified in Figure 2A-C. The contrast in the binding of actin filaments to HMM (in the presence of ATP) on a micropatterned surface is most clearly demonstrated in Figure 2A. This Figure shows HMM-attached APh-labeled actin filaments that had been added in high concentration (1 µM actin; 20% APh-labeled). The high-quality function on TMCS is more evident in Figure 2B. This Figure shows the sum of 90 black and white images of the same area as in Figure 2A after switching to a filter set for the observation of RhPh-labeled actin filaments (added at 2 nM). The paths traced out by these filaments are shown with time markers every 12 s, indicating average velocities of 3 µm s-1 (18 °C). It can also be seen that the filaments did not slide outside the TMCS-derivatized area. Similar results were observed for the nanopatterned surface in Figure 2C. This Figure shows the complete selectivity of function in a40 solution with actin filament sliding on nanosized TMCS tracks but not on the surrounding SiO2 surfaces. Conditions for Complete Motility Selectivity between TMCS and SiO2. Complete motility contrast between TMCS and SiO2 in the absence of methylcelluose was also observed at ionic strengths other than 40 mM and HMM incubation concentrations different from those used in Figure 1. This is illustrated in Figure 3. Here it can be seen that at ionic strengths of 20-50 mM there was no actin filament sliding on SiO2 when HMM was added at concentrations below 150 µg mL-1. The lack of motility on SiO2 was extended to the highest HMM incubation concentration (200 µg mL-1) for ionic strengths of 40 and 50 mM. At 20-40 mM ionic strength and HMM incubation concentrations g30 µg mL-1, there was high-quality motility on TMCS and thus complete motility contrast between SiO2 and TMCS. It can also be seen in Figure 3 that motility contrast was

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Figure 1. Actomyosin motor function on TMCS-derivatized surfaces and on pure SiO2. (A) Summary of results showing the sliding velocity (filled bars) and fraction of motile filaments (striped bars) on vapor-deposited TMCS (CVD-TMCS) and on SiO2 in aMC130 (black) and a40 (grey) solutions. The number of replicate experiments (n) is given in parentheses. Data normalized to values on control nitrocellulose surfaces in each experiment. (Fraction of motile filaments and velocity on nitrocellulose: 0.81 ( 0.01 and 8.42 ( 0.86 µm s-1 in aMC130 assay solution; 0.82 ( 0.01 and 5.72 ( 0.80 µm s-1 in a40 assay solution.) Fraction of motile filaments estimated only in experiments with automatic tracking. No motility was observed on SiO2 using assay solution a40 (11 experiments). Temperature: 28.3-31.2 °C except for one experiment on SiO2 in aMC130 solution (23 °C) and two experiments on TMCS in a40 solution (24 °C). (B) Left panel: Filament (arrow) in aMC 130 assay solution sliding on a micropatterned surface with a TMCS-derivatized circle (delineated by a dashed white line) surrounded by pure SiO2. Sum of 50 fluorescence image frames from an in vitro motility assay showing trajectories followed by HMM-propelled actin filaments. Position of filaments indicated every 3.4 s by bright time markers on trajectories. Scale bar 10 µm. Note the longer distance between time markers on TMCS indicating higher velocity. Inset: bright-field microscopy image (image size 85 × 85 µm2) in reflected light showing water vapor condensed on the micropatterned surface. Right panel: Frame-to-frame velocity of one filament (arrow, left panel) crossing the SiO2/TMCS border. (C) Dependence on contact angle of sliding velocity on glass (open squares) and on SiO2 (circles). Advancing or static contact angle (open symbols) or receding contact angle (filled circles; SiO2 only). Assay solution aMC130. Velocity data normalized to velocity on control nitrocellulose or TMCS surfaces. Each data point represents a mean ( SE of the velocity from 22 to 119 filament paths. The solid line is fitted to data for glass and SiO2 by linear regression. (D) Time dependence of sliding velocity on a micropatterned surface after the first incubation in aMC130 solution on SiO2 (open circles) or TMCS-derivatized areas (filled circles). The surface was incubated with aMC130 solution from time zero up to 50 min. Here the surface was rinsed several times with wash solution (three arrows), followed by more than 60 min of incubation in a40 solution. This incubation was terminated by several new rinses with wash solution (after about 130 min; arrows), followed by the new addition of aMC130 solution.

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Figure 3. Actin filament velocities on bare SiO2 (open symbols; data partially replotted from ref 25), vapor-deposited TMCS on SiO2 (black filled symbols), and liquid-deposited TMCS on SiO2 (grey filled symbols and lines; data replotted from ref 25). Different symbol shapes indicate different assay solutions; a50, square; a40, diamond; a30, triangle; a20, circle. The number of filament paths used for velocity calculations, if less than 20, is given in parentheses. Note the few filaments in all experiments on silicon dioxide and at the highest ionic strength on TMCS. Also note that the data points for SiO2 at 200 µg/mL HMM at zero velocity include a20, a30, and a40 assay solutions. Furthermore, the data points for SiO2 at 120130 µg/mL HMM at zero velocity represent the results of eight different surfaces on five experimental occasions using a40 assay solution. The lines connect data obtained during a given experimental occasion to simplify viewing. Figure 2. Motility selectivity in a40 solution demonstrated on microand nanopatterned surfaces. (A) Filament sliding in a40 assay solution on a micropatterned surface with the TMCS-derivatized area to the left (bright). Surface preincubated with HMM at a concentration of 130 µg mL-1. Fluorescence image of APh-labeled blocking actin (1 µM, 20% labeled, 0.5 s exposure) showing the selective localization of the actin filaments on the TMCS-derivatized area. Scale bar 15 µm, temperature 18 °C. (B) Same surface as in part A but after exchange with the fluorescence filter set for the observation of RhPhlabeled actin filaments. Sum of 90 fluorescence images (0.5 s exposures) of RhPh-labeled actin filaments showing filament trajectories. Positions of filaments indicated by bright time markers every 12 s. Border between TMCS and SiO2 indicated by dashed lines. (C) Trajectories of HMM-propelled actin filaments on nanosized TMCS tracks (left; width 100-400 nm) and the TMCS loading zone (right). TMCS areas surrounded by bare SiO2 where no sliding filaments were observed. Sum of 82 background-subtracted images (exposure 0.2 s, assay solution a40). Scale bar 10 µm, temperature 23 °C.

observed for similar conditions whether TMCS was deposited from the vapor phase or from solution (tested on separate homogeneous surfaces; some of the data are from ref 25). As mentioned above, complete motility contrast (no motility on SiO2) was only occasionally observed in the presence of methylcellulose in the assay solution. Although not systematically studied, motility on SiO2 in the presence of methylcellulose was also demonstrated under other experimental conditions (lower HMM density and 40 mM ionic strength) than those in Figure 1A. Mechanism for Motility Selectivity between TMCS and SiO2. One possible explanation for the difference in actomyosin function between SiO2/glass and TMCS could be a difference in HMM surface density. Below, we describe tests of this hypothesis using different experimental systems. For practical reasons, the tests were performed either on liquiddeposited TMCS on glass (where a large number of flow cells were required) or on liquid-deposited TMCS on fused silica (silica glass, SiO2; where UV transmission was required). In all cases, corresponding nonderivatized surfaces were used as controls. A. K/EDTA-ATPase Measurements. As in several earlier studies (e.g., refs 39 and 42), the myosin head density was first assessed

Figure 4. HMM density plotted against HMM incubation concentration on different surface chemistries. The circles illustrate the densities of catalytically active HMM molecules on glass (open circles) and TMCS-derivatized glass (closed circles) as measured by K/EDTA-ATPase activity. Three batches of TMCS-derivatized glass surfaces used except at 120 µg mL-1 (five batches) and 200 µg mL-1 (two batches). For each batch, data were pooled from two to six flow cells for each HMM incubation concentration and normalized to values obtained on glass at 120 µg mL-1 in the same experiment. Lines represent Boltzmann sigmoidal functions fitted to the data (TMCS: full line; glass: dashed line).

by ATPase (here, K/EDTA-ATPase) measurements in flow cells where HMM had been adsorbed either on nonfunctionalized or TMCS-derivatized glass cover slips. At an HMM incubation concentration of 120 µg mL-1, comparison with ATPase activity in solution in two experiments suggested that the surface density of HMM was 6700 ( 380 molecules µm-2 on TMCS (compared to 5800 ( 310 molecules µm-2; n ) 2 on nitrocellulose). In Figure 4, it can be seen that for both TMCS and glass the saturation of HMM binding to the surface occurred at HMM incubation concentrations between 60 and 100 µg mL-1. A significant difference (p < 0.05) in the density of catalytically active HMM molecules between TMCS and glass could be verified at an incubation concentration of 120 µg mL-1. Here, the density was 26 ( 5% (n ) 5; paired observations) lower on glass than on TMCS. (42) Uyeda, T. Q.; Warrick, H. M.; Kron, S. J.; Spudich, J. A. Nature 1991, 352, 307-311.

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Figure 5. Fluorescence spectroscopy data showing (A) HMM adsorption to silica glass (dashed line) and TMCS (full line) and subsequently in the same experiment (B) the binding of APh-labeled actin filaments to the HMM on silica glass (dashed line) and TMCS (full line). The data in A and B were obtained with evanescent wave excitation. (C) HMM incubation and adsorption to silica glass and TMCS recorded using transmitted light. Note the large contribution of HMM in the bulk during the adsorption phase. Straight lines during the incubation period indicate the fluorescence intensity attributed to HMM on the surface. In A and C, HMM was added at 130 µg/mL at the first arrow, followed by rinsing with wash solution at the second arrow. In B, a mixture of nonlabeled and APh-labeled actin was added (in the absence of ATP) at a concentration of 1 µM, followed by rinsing with wash solution at the second arrow and with a40 solution at the third arrow. In A and C, the proteins were subjected to excitation at 280 nm (either evanescent wave or transmitted light), and fluorescence emission was measured at 335 nm. The infusion of APh actin (B) was monitored by evanescent wave excitation at 495 nm and the recording of emission at 519 nm. The same integration time (0.5 s) and sampling rate (0.5 s-1) were used in all measurements. (D) Emission spectrum of HMM adsorbed to TMCS (full line) or silica glass (dashed line) after background subtraction and normalization to the peak value on each surface. Note the complete superposition of spectra on TMCS and silica glass. Excitation at 280 nm is by transmitted light.

B. HMM Depletion upon Flow Cell Incubation. The HMM content of the incubation solution was measured before and after 2 min of incubation of flow cells. By this approach, we obtained a measure of HMM density on TMCS relative to that of SiO2 that was independent of any differences in myosin ATPase activity on the two surfaces. In contrast to the ATPase measurements (see above), the measurements of HMM depletion suggested a 24.6 ( 4.5% (n ) 3; p < 0.05) higher HMM density on SiO2 than on TMCS. C. Fluorescence Spectroscopy. The HMM surface density and the density of HMM molecules with actin binding capability were also assessed using fluorescence spectroscopy. In these experiments, the TMCS-derivatized silica glass surfaces exhibited an advancing contact angle of 67 ( 4° (n ) 4 surfaces) compared to 19 ( 5° (n ) 4) for nonsilanized silica glass. The surfaces were first incubated with HMM (130 µg mL-1), followed by BSA (1 mg mL-1), and finally with fluorescently labeled actin filaments. The data in Figure 5A and B were obtained using evanescent wave excitation. It can be seen in Figure 5A that the fluorescence intensity, attributed to surface-adsorbed HMM, was higher on pure silica glass than on TMCS (by 24 and 40% in two experiments) both during HMM incubation and after rinsing

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with the wash solution. These differences are in good agreement with those (30.4 ( 10.1%; range: 15-60%; p ) 0.057) observed using the same setup in four other experiments but with transmitted light rather than evanescent wave excitation (Figure 5C). In Figure 5C, it can be seen that the fluorescence intensity attributed to HMM in solution during the incubation phase was similar for TMCS and silica glass surfaces. This argues against any trivial reason for lower intensity on TMCS such as increased absorption of the exciting or emitted light by this surface. The differences in fluorescence intensity between the HMM adsorbed to TMCS and silica glass were not accompanied by any differences in the shape of the HMM emission spectrum (Figure 5D). Incubation with RhPh actin at low concentration (2 nM; not shown) followed by APh actin at high concentration (1 µM actin, 40 nM APh labeled; Figure 5B) both showed less binding of actin to the HMM on pure silica glass than to that on TMCS. In six experiments (pooled data from transmitted light and evanescent wave excitation), the fluorescence intensity due to APh actin was 78 ( 3% (p < 0.001) lower on pure silica glass than on TMCS with both surfaces preincubated with HMM as described above. Rinsing with ATP-containing solution (a40) reduced the APh fluorescence to the baseline on silica glass but produced only a small reduction on TMCS (Figure 5B). In one of the fluorescence spectroscopy experiments, a motility assay was performed on the TIRF slide after 2 h of measurement, confirming that actomyosin function was maintained on TMCS under these conditions. (For details, see Supporting Information.) Filament Length and Sliding Velocity. The above results suggest larger HMM density on SiO2 than on TMCS and only small differences between TMCS and SiO2 in the density of myosin heads with catalytic activity. This cannot explain the complete motility contrast between TMCS and SiO2 in a40 solution (Discussion). However, the above measurements of HMM density do not assess what fraction of the myosin heads actually bind actin filaments during myosin-induced sliding. To get access to this fraction, we fitted eq 1 to plots of sliding velocity versus actin filament length to obtain the quantity Fd. This quantity represents the number of myosin heads per actin filament length participating in force generation. On TMCS, we assume that the value is 13 000 × 0.03 ) 390 µm-1 (see Methods; F approximated from K/EDTA-ATPase data), and the parameter η is here taken to be 1 immediately after incubation with assay solution. In Figure 6A, the sliding velocity is plotted against the actin filament length for filaments that were sliding on both TMCS and SiO2 on a micropatterned surface. The data were collected within 5 min after the first infusion of assay solution. It can be seen that the results for TMCS are well accounted for by using f ) 0.05 and Fd ) 390 µm-1 (see above and the legend of Figure 6). The best fit of the model to the SiO2 data was obtained with a considerably lower value of Fd (87 ( 22 µm-1) and a slightly lower value of η (0.95 ( 0.03). The inset of Figure 6A shows the ratio between the velocity on SiO2 and TMCS for each given filament that was sliding on both surface chemistries. The full line in the inset represents the ratio between the equations represented by the full lines in the main figure in Figure 6A (Fd ) 390 µm-1 on TMCS and 87 µm-1 on SiO2). The dashed line in the inset of Figure 6A represents the ratio for the case in which Fd is the same on TMCS and SiO2. It can be seen that the data, despite the scatter, are clearly consistent with a lower fraction of force producing myosin heads on SiO2 (full line in inset) whereas they would be difficult to reconcile with a similar fraction on the two surface chemistries (dashed line).

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Figure 6. Sliding velocity plotted against actin filament length. (A) Velocity data for filaments moving on both SiO2 (open symbols) and TMCS areas (filled symbols) on a micropatterned surface. Data obtained within 5 min after the first addition of assay solution. The lower solid line represents the fitting of eq 1 to the SiO2 data, giving a values of Fd ) 87 ( 22 µm-1 and η ) 0.95 ( 0.03. Data plotting is limited to actin filament lengths below 5 µm, but five additional data points for filaments of lengths up to 12 µm are included in the curve fitting. Equation 1 for TMCS data is plotted using Fd ) 390 µm-1 and η ) 1 (see the text). The inset shows the ratio between the sliding velocity for each given filament on SiO2 and TMCS plotted against filament length. The solid line represents the ratio between the two equations illustrated by the two solid lines in the main figure. The dashed line represents this ratio if the quantity Fd would have been equal on TMCS and SiO2. (B) Plot of sliding velocity against filament length for same surface as in A but 2 h after the first addition of assay solution and after two exchanges of assay solution. The meanings of symbols and lines are the same as in A, but eq 1 (full lines) was fitted to the data for both TMCS and SiO2. The best fit on TMCS was obtained with Fd ) 39 ( 5 µm-1 and η ) 0.91 ( 0.03 whereas the best fit on SiO2 was obtained with Fd ) 20 ( 1 µm-1 and η ) 0.74 ( 0.03 (Inset) Same type of plot as in A and the same meanings of symbols and lines. (C) Velocity data obtained from filaments moving on separate nonpatterned surfaces. Filled circles represent filaments moving on a TMCS surface (contact angle 79°), and open circles represent filaments moving on a piranha-cleaned SiO2 surface (contact angle 32°). The lower solid line represents the fitting of eq 1 to the SiO2 data (Fd ) 43 ( 6 µm-1 and η ) 0.56 ( 0.02). Equation 1 for the TMCS data is plotted using Fd ) 390 µm-1 and η ) 1 as in Figure 7A. Data plotting is limited to actin filament lengths below 5 µm, but three additional data points for filaments of lengths up to 11 µm are included in the curve fitting. The assay solution was aMC130 in A-C. Fittings of eq 1 to data are given with 95% confidence limits (dashed lines).

In Figure 6B, data for the same surface as in Figure 6A were collected about 2 h after the adsorption of HMM to the surface and after several exchanges of assay solution. The best fit to these data was obtained with Fd ) 39 ( 5 µm-1 and η ) 0.91 ( 0.027 for TMCS and Fd ) 20.3 ( 1.2 µm-1 and η ) 0.74 ( 0.03 for SiO2. Similar to the situation immediately after the infusion of assay solution (Figure 6A), these data suggest that fewer myosin heads contribute to the sliding motion on SiO2 than on TMCS. Furthermore, they suggest that the number of available myosin heads has decreased with time on both surface chemistries. Differences between TMCS and SiO2, which are qualitatively similar to those in Figure 6A and B for the quantities Fd and η, were found in one more experiment using a micropatterned surface and in three experiments using nonpatterned surfaces. In two of the experiments with nonpatterned surfaces, the difference between SiO2 and TMCS in average sliding velocity was accentuated by piranha cleaning of the SiO2 surface, resulting in a lower contact angle (about 30°). The results from one of these experiments are illustrated in Figure 6C. Fitting of eq 1 to the data in this Figure suggests that Fd was 43 ( 5.9 µm-1, which is considerably lower than on TMCS (390 µm-1). Pooling data from five different surface-protein combinations (irrespec-

tive of contact angle on SiO2 and of time of observation after the first incubation in assay solution) gave values of Fd that were 44-90% lower on SiO2 than on TMCS with an average reduction of 62 ( 7% (p < 0.001). This reduction was significantly larger (p < 0.001) than that suggested by the K/EDTA-ATPase measurements (see above). Motility-Suppressing PMMA Surfaces. We treated nonpatterned PMMA surfaces in a way similar to that previously26 used in the fabrication of nanostructured surfaces. An important step in the treatment was oxygen plasma ashing.26 Before plasma treatment, PMMA exhibited advancing and receding contact angles with water of 77 ( 1 and 56 ( 2°, respectively (n ) 2). The quality of actomyosin motility (sliding speed and fraction of motile filaments) under these conditions was similar to that on nitrocellulose. After treatment, as during nanostructuring, there was a reduction of the advancing and receding contact angles to 62 ( 2 and 21 ( 3°, respectively (n ) 4). These changes were associated with a loss of actomyosin motility in the absence of methylcellulose. The addition of methylcellulose to the assay solution restored motility. In general agreement with the data for glass and SiO2 (Figure 1), the motility quality was, however, low with low

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velocity and a low fraction of motile filaments (HMM incubation concentration of 200 µg mL-1).

Discussion Glass, SiO2, and the TMCS Deposition Method. In analyzing the mechanisms for the motility contrast between TMCS and SiO2/glass, we made the following assumptions based on results in this article and earlier work.25 First, liquid-deposited TMCS on glass/SiO2 and vapor-deposited TMCS on SiO2 wafers are regarded as equivalent both with respect to HMM binding and actomyosin motility. Second, silicon dioxide on Si/SiO2 wafers and silicon dioxide-like surfaces (e.g., glass) are assumed to behave similarly both with respect to HMM binding and actomyosin function. In regard to equivalence between liquid-deposited and vapordeposited TMCS, this is consistent with the similarities in advancing and receding contact angles between the surfaces (∼80 and 70°, respectively; see Results and ref 25) and the great similarities in the quality of motility. Thus, on both vapordeposited and liquid-deposited TMCS the fraction of motile filaments and the sliding velocity were similar to, and often better than, those on nitrocellulose (cf. Figure 1 and ref 25). Furthermore, high-quality motility was observed for similar ionic strengths and HMM incubation concentrations in the absence of methylcellulose in the assay solution (Figure 3). The assumption that glass and SiO2 behave similarly to underlying substrates in the motility assay is also supported by our experimental evidence. Thus, neither on glass nor on SiO2 was there any motility in the absence of methylcellulose in the assay solution. Furthermore, the motility quality was poor in the presence of methylcellulose (low velocity; low fraction of motile filaments) for glass and SiO2 surfaces with contact angles in a similar range (e.g., Figure 1C). In accordance with the functional similarities between glass and Si/SiO2 wafers, SiO2 is the dominant chemical constituent (64% of total mass) of the borosilicate cover glasses used (Schott D263M). This similarity in chemical composition is consistent with the similarity in contact angle between the two surfaces provided that they are cleaned in a similar way (e.g., cleaning of both surfaces using piranha solution). It has also been shown that glass fibers with a wide range of elemental compositions and SiO2 content (from about 50 to >99%) all have a negative charge at pH 7-8 due to the dissociation of protons from surface hydroxyl groups.27 In view of the above evidence, we assume that the HMM binding as well as the actomyosin interactions are similar on glass and SiO2. On SiO2 surfaces in the form of fused silica (silica glass), a more extensive cleaning procedure (involving piranha solution) was required for effective TMCS derivatization from the liquid phase. With this cleaning procedure, the TMCS surfaces exhibited an average contact angle that was almost as high as that on TMCS glass. Because the piranha-cleaned silica glass surfaces had a low contact angle (50° were associated with optimal actomyosin function. Although not studied in detail, it is likely that there is also an upper limit for suitable contact angles. Thus, Jaber et al.,45 without giving contact angle data, presented results showing a lack of motility on a “hydrophobic” PEBSS [poly(styrene sulfonate)block-poly(ethylene-ran-butylene)-block-poly(styrene sulfonate)] surface. In contrast to the high-quality motility on TMCS and nitrocellulose, there was no motility in the absence of methylcellulose in our experiments on SiO2, glass, and oxygen-plasmatreated PMMA surfaces. Furthermore, on SiO2 and glass the sliding velocity in the presence of methylcellulose was reduced with decreases in the advancing contact angle below 70° (and the receding contact angle below 50°). The studied hydrophilic surfaces (SiO2, glass, and oxygen-plasma-treated PMMA) are all likely to exhibit an excess of negatively charged groups at the pH used (see above and ref 28), and none of them supported actomyosin motility in the absence of methylcellulose. It is interesting that the lack of motility, in the absence of methylcellulose, was also observed for negatively charged polyelectrolyte multilayers and monolayers.45 In addition, just as in the present work, significant binding of both HMM and actin to the surface was observed without motility. In contrast to the effect of negatively charged surfaces, good motility was observed by Jaber et al.45 on positively charged polyelectrolyte multilayers and monolayers. The differences in motility quality between hydrophilic surfaces with excess positive and negative charge are consistent with a model for the motility contrast between TMCS and SiO2/glass proposed below. Mechanism of Motility Contrast between TMCS and SiO2/ Glass. Our experimental results (HMM depletion upon incubation and fluorescence spectroscopy) suggest that the HMM density was higher on SiO2/glass than on TMCS-derivatized surfaces despite poor or very poor motility. The slightly lower average ATPase activity of the HMM molecules (Figure 4) on glass is not sufficient to explain the large motility contrast between TMCS and glass/SiO2 in a40 assay solution. Thus, the data in Figure 3 show motility on TMCS at HMM incubation concentrations down to 30 µg mL-1 corresponding to K/EDTA-ATPase activity that is about 60% lower than on TMCS at saturating HMM concentrations (Figure 4). This should be compared to the ATPase activity on glass at saturation that was only 27% lower than on TMCS but with complete motility contrast. (44) Nicolau, D. V.; Suzuki, H.; Mashiko, S.; Taguchi, T.; Yoshikawa, S. Biophys. J. 1999, 77, 1126-1134. (45) Jaber, J. A.; Chase, P. B.; Schlenoff, J. B. Nano Lett. 2003, 3, 1505-1509.

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A motility contrast in the absence of major differences in HMM density or ATPase activity suggest that HMM may bind and propel actin considerably less effectively on SiO2/glass than on TMCS. The idea is in accordance with the fluorescensce spectroscopy data showing >60%54 lower actin binding to HMM on SiO2 than on TMCS. The values of the product Fd obtained from the velocity-length plots (Figure 6) are also consistent with a lower density (by about 60%) of actin propelling myosin heads on SiO2 than on TMCS. Also, the lower value of the parameter η in the velocity-length plots on SiO2 is consistent with a substantially lower density of actin propelling HMM molecules on this surface.8 Taken together, the above discussion suggests that the main mechanism underlying actomyosin motility selectivity between SiO2/glass and TMCS is a different mode of adsorption resulting in a different function of HMM on the different surfaces. The lack of spectral shifts (Figure 5D) indicates that this occurs without major changes in myosin head structure. The experimental results are consistent with a simplified model with two populations of HMM molecules: one with actin-binding and actin-propelling properties (HMMC; dominating on TMCS) and the other with poor or no actin binding and with a lower K/EDTA-ATPase activity (HMMN; seen only on SiO2/glass). In a structural model, the HMMC molecules may correspond to those found by Toyoshima30 to attach to nitrocellulose surfaces via a single attachment point close to the most C-terminal part (near the HMM-LMM junction). Because this part is flexible, it is expected to bind not only to TMCS but also to hydrophilic SiO2 and glass surfaces even if these have opposite charge (cf. ref 46). The HMMN molecules, however, may correspond to those, in the work of Toyoshima, that appeared to be tethered to nitrocellulose surfaces via the actual motor domain of S1 with resulting poor actin translating capability. The binding of HMM in this configuration may be promoted on negatively charged surfaces46 (e.g., SiO2/glass) because the myosin head has excess positive charge (Swiss-Prot entry Q28641 and ref 47). The above model implies that there may be a different average distance between the myosin heads and the surface on TMCS and SiO2 because several of the myosin heads are assumed to be tethered on the latter surface. One might then expect an increased difference in HMM fluorescence between the two surfaces when using evanescent wave excitation (rather than transmitted light). However, this effect would be small (