| 1. |
- Mansson, A, et al.
(författare)
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Actin-based molecular motors for cargo transportation in nanotechnology - Potentials and challenges
- 2005
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Ingår i: IEEE Transactions on Advanced Packaging. - 1521-3323. ; 28:4, s. 547-555
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Tidskriftsartikel (refereegranskat)abstract
- Here, we review the use of actin-based motors, (myosins; e.g., the molecular motor of muscle) in. nanotechnology. The review starts from the viewpoints of the molecular motors as being important devices responsible of cargo transportation in the cell and end in discussions about their employment in nanotechnological applications. First, we describe basic biophysics of the myosin motors with focus on their involvement in cargo transportation in the living cell, leading us over into a discussion about in vitro motility assays. These are biological test systems where the myosin-induced translocation of actin filaments is studied on an artificial surface outside the cell. Then follows a review about modified motility assays for production of ordered motion. Here, we discuss ours and others' work with regards to making micro- and nanostructured surfaces and channels where the position and direction of movement produced by molecular motors is controlled. In this section, we consider the role of the channel size in promoting unidirectional myosin-induced motion of actin filaments. Furthermore, we consider the usefulness of surface modifications, e.g., various silanization procedures in order to promote and hinder molecular motility, respectively. Particularly, we describe our latest test system being both morphologically and chemically nanostructured giving us unsurpassed possibilities to perform functional studies as well as extremely good spatio-temporal control. Then follows a section about nanotechnological cargo transportation systems based on the actomyosin motor system. For instance, we present results of attaching fluorescent quantum dots as cargoes to the actin filaments. In this section, we also discuss the possibilities of having cargo attachment and detachment being performed on demand. Finally, we consider the usefulness of molecular motors for lab-on-a-chip applications and the requirements for incorporating these motors in commercially viable devices. In this context, the significant potential of the actomyosin motor system to overcome traditional limitations of micro- and nanofluidics is stressed.
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| 2. |
- Mansson, A, et al.
(författare)
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In vitro sliding of actin filaments labelled with single quantum dots
- 2004
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 314:2, s. 529-534
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Tidskriftsartikel (refereegranskat)abstract
- We recently refined the in vitro motility assay for studies of actomyosin function to achieve rectified myosin induced sliding of actin filaments. This paves the way, both for detailed functional studies of actomyosin and for nanotechnological applications. In the latter applications it would be desirable to use actin filaments for transportation of cargoes (e.g., enzymes) between different predetermined locations on a chip. We here describe how single quantum dot labelling of isolated actin filaments simultaneously provides handles for cargo attachment and bright and photostable fluorescence labels facilitating cargo detection and filament tracking. Labelling was achieved with preserved actomyosin function using streptavidin-coated CdSe quantum dots (Qdots). These nanocrystals have several unique physical properties and the present work describes their first use for functional studies of isolated proteins outside the cell. The results, in addition to the nanotechnology developments, open for new types of in vitro assays of isolated biomolecules. (C) 2003 Elsevier Inc. All rights reserved.
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| 3. |
- Bunk, Richard, et al.
(författare)
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Actomyosin motility on nanostructured surfaces
- 2003
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Ingår i: Biochemical and Biophysical Research Communications. - 1090-2104. ; 301:3, s. 783-788
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Tidskriftsartikel (refereegranskat)abstract
- We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200 nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.
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| 4. |
- Bunk, Richard, et al.
(författare)
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Guiding molecular motors with nano-imprinted structures
- 2005
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Ingår i: Japanese Journal of Applied Physics. - 0021-4922. ; 44:5A, s. 3337-3340
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Tidskriftsartikel (refereegranskat)abstract
- This work, for the first time, demonstrates that nano-imprinted samples, with 100 nm wide polymer lines, can act as guides for molecular motors consisting of motor proteins actin and myosin. The motor protein function was characterized using fluorescence microscopy and compared to actomyosin motility on non-structured nitrocellulose surfaces. Our results open for further use of the nano-imprint technique in the production of disposable chips for bio-nanotechnological applications and miniaturized biological test systems. We discuss how the nano-imprinted motor protein assay system may be optimized and also how it compares to previously tested assay systems involving low-resolution UV-lithography and low throughput but high-resolution electron beam lithography.
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| 5. |
- Bunk, Richard, et al.
(författare)
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Guiding motor-propelled molecules with nanoscale precision through silanized bi-channel structures
- 2005
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Ingår i: Nanotechnology. - : IOP Publishing. - 0957-4484 .- 1361-6528. ; 16:6, s. 710-717
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Tidskriftsartikel (refereegranskat)abstract
- We report on the design and fabrication of a channel structure for high precision guidance and achieving excellent confinement properties for motor-propelled molecular shuttles. The techniques used to manufacture the channel structure are mainly e-beam lithography and selective monolayer functionalization. The structure consists of two lateral layers of concentric channels on a SiO2 surface made biocompatible with the molecular motors. The quality and advantages of the design are demonstrated by experiments using the motor proteins actin and myosin. The special channel geometry leads to stable biochemical conditions with full motor protein functionality. ATP is sufficiently supplied to all parts of the structure by dedicated service channels, as is the venting of ADP and P-i (inorganic phosphorus). Channels of different widths (100-700 nm) and shapes are fabricated and measurements made on them.
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| 6. |
- Bunk, Richard, et al.
(författare)
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Towards a 'nano-traffic' system powered by molecular motors
- 2003
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Ingår i: Microelectronic Engineering. - 1873-5568. ; 67-8, s. 899-904
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Tidskriftsartikel (refereegranskat)abstract
- In this work, we reconstructed in vitro the behavior of two motor proteins-myosin and actin-responsible for the mechanical action of muscle cells. By transferring this in vivo system to an artificial environment, we were able to study the interaction between the proteins in more detail, as well as investigating the central mechanism of force production. Nm-patterning by e-beam lithography (EBL) could restore parts of the in vivo protein order, essential for potential nanotechnological applications. Much work was put into establishing the necessary compatibility between the biological and nano-lithographical processes. A range of EBL-resists were tested for protein compatibility. One particular kind (MRL-6000.1XP) supported good actin filament motility, while another (PMMA-950) behaved in the opposite way. Taking advantage of these findings, nm-sized lines were created in a double-layer structure of the two resists. The lines were found to act as binding sites for myosin, and as rectifying guides for the linearized motion of actin filaments. Velocities around 5 mum/s were measured. (C) 2003 Elsevier Science B.V. All rights reserved.
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| 7. |
- 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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| 8. |
- 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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| 9. |
- Sundberg, M, et al.
(författare)
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Silanized surfaces for in vitro studies of actomyosin function and nanotechnology applications
- 2003
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 323:1, s. 127-138
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Tidskriftsartikel (refereegranskat)abstract
- We have previously shown that selective heavy meromyosin (HMM) adsorption to predefined regions of nanostructured polymer resist surfaces may be used to produce a nanostructured in vitro motility assay. However, actomyosin function was of lower quality than on conventional nitrocellulose films. We have therefore studied actomyosin function on differently derivatized glass surfaces with the aim to find a substitute for the polymer resists. We have found that surfaces derivatized with trimethylchlorosilane (TMCS) were superior to all other surfaces tested, including nitrocellulose. High-quality actin filament motility was observed up to 6 days after incubation with HMM and the fraction of motile actin filaments and the velocity of smooth sliding were generally higher on TMCS than on nitrocellulose. The actomyosin function on TMCS-derivatized glass and nitrocellulose is considered in relation to roughness and hydrophobicity of these surfaces. The results suggest that TMCS is an ideal substitute for polymer resists in the nanostructured in vitro motility assay. Furthermore, TMCS derivatized glass also seems to offer several advantages over nitrocellulose for HMM adsorption in the ordinary in vitro motility assay. (C) 2003 Elsevier Inc. All rights reserved.
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