Biomedical subjects

Martina Balaz

Publications and source records attributed to Martina Balaz.

3 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↗

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↗