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- Sundberg, Mark, et al.
(författare)
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Actin filament guidance on a chip: Toward high-throughput assays and lab-on-a-chip applications
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:17, s. 7286-7295
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Tidskriftsartikel (refereegranskat)abstract
- Biological molecular motors that are constrained so that function is effectively limited to predefined nanosized tracks may be used as molecular shuttles in nanotechnological applications. For these applications and in high-throughput functional assays (e. g., drug screening), it is important that the motors propel their cytoskeletal filaments unidirectionally along the tracks with a minimal number of escape events. We here analyze the requirements for achieving this for actin filaments that are propelled by myosin II motor fragments (heavy meromyosin; HMM). First, we tested the guidance of HMM-propelled actin filaments along chemically defined borders. Here, trimethylchlorosilane (TMCS)-derivatized areas with high-quality HMM function were surrounded by SiO2 domains where HMM did not bind actin. Guidance along the TMCS-SiO2 border was almost 100% for filament approach angles between 0 and 20 degrees but only about 10% at approach angles near 90 degrees. A model (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) accounted for essential aspects of the data and also correctly predicted a more efficient guidance of actin filaments than previously shown for kinesin- propelled microtubules. Despite the efficient guidance at low approach angles, nanosized (< 700 nm wide) TMCS tracks surrounded by SiO2 were not effective in guiding actin filaments. Neither was there complete guidance along nanosized tracks that were surrounded by topographical barriers (walls and roof partially covering the track) unless there was also chemically based selectivity between the tracks and surroundings. In the latter case, with dually defined tracks, there was close to 100% guidance. A combined experimental and theoretical analysis, using tracks of the latter type, suggested that a track width of less than about 200-300 nm is sufficient at a high HMM surface density to achieve unidirectional sliding of actin filaments. In accord with these results, we demonstrate the long- term trapping of actin filaments on a closed-loop track (width < 250 nm). The results are discussed in relation to lab-on-a-chip applications and nanotechnology-assisted assays of actomyosin function.
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| 2. |
- Sundberg, Mark, et al.
(författare)
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Selective spatial localization of actomyosin motor function by chemical surface patterning
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:17, s. 7302-7312
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Tidskriftsartikel (refereegranskat)abstract
- 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 degrees (advancing contact angles for water droplets). The corresponding contact angle of CVD-TMCS was 81 degrees. 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 mu g 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.
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