Biomedical subjects

Alf Månsson

Publications and source records attributed to Alf Månsson.

6 recordsLinked to original sources

PubMed2006

Selective spatial localization of actomyosin motor function by chemical surface patterning.

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, SiO(2), 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/SiO(2) and on TMCS, but the velocity was higher on TMCS. This difference in velocity increased with decreasing contact angles of the glass and SiO(2) 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/SiO(2). This observation was independent of the contact angle of the glass/SiO(2) surfaces and of HMM incubation concentrations (30-150 microg mL(-)(1)) and ionic strengths of the assay solution (20-50 mM). Complete motility selectivity between TMCS and SiO(2) 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 SiO(2)/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 SiO(2)/glass.

Actomyosin↗

PubMed2006

Actin filament guidance on a chip: toward high-throughput assays and lab-on-a-chip applications.

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 SiO(2) domains where HMM did not bind actin. Guidance along the TMCS-SiO(2) 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 SiO(2) 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.

Actin Cytoskeleton↗

PubMed2005

Detection of small differences in actomyosin function using actin labeled with different phalloidin conjugates.

This study shows that there is only a negligible difference in actomyosin function in the in vitro motility assay among actin filaments labeled with Rhodamine phalloidin (RhPh), Alexa-488 phalloidin (APh), and biotin-XX phalloidin (BPh). Similar results were obtained at varying ionic strengths (0.02-0.13 M), in the presence of imidazole or 3-[N-morpholino]propanesulfonic acid (MOPS) buffer, and at varying MgATP concentrations (0.1-3 mM). If RhPh- and APh-labeled filaments were studied in a given flow cell, there was minimal variability in sliding velocity between the fluorophores (standard deviation of 3% of the absolute sliding velocity). The variability was considerably smaller than that between flow cells, allowing us to use dual labeling of different actin types and then apply analysis of variance to detect minor functional differences between them. Using this method, we could statistically verify a 4% difference (P<0.001) in sliding velocity (3mM Mg ATP) between cardiac and skeletal muscle actin. Suggested improvements of the method would readily allow the detection of even smaller differences. We discuss implications of the results for nanotechnological applications, understanding actomyosin function, and reducing experimental costs and the use of laboratory animals.

Actins↗

PubMed2004

In vitro sliding of actin filaments labelled with single quantum dots.

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.

Actin Cytoskeleton↗

PubMed2003

Silanized surfaces for in vitro studies of actomyosin function and nanotechnology applications.

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.

Actomyosin↗

PubMed2003

Actomyosin motility on nanostructured surfaces.

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 (100-200nm 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.

Actin Cytoskeleton↗