Murat Kekic

Manipulation of the motility of protein molecular motors on microfabricated substrates

Heavy meromyosin (HMM), a proteolytically cleaved derivative of myosin has previously been shown to interact with actin in well-established in vitro motility assays on nitrocellulose surfaces. In this study, the assays were conducted to demonstrate that the motility of actin filaments is confined in the micron-sized channels fabricated via laser ablation in a layer of the photosensitive resist polymer O-acryloyloxime acetophenone oxime (AAPO). A solution containing myosin labeled with fluorophore 5-iodoacetamidofluorescein (5-IAF) was applied to the microfabricated AAPO surface and shown to bind specifically to the micron-size channels. In the motility assay, HMM, rhodamine-phalloidin labeled actin and ATP were sequentially added and the movement of the actin filaments was observed by fluorescence microscopy and recorded with a CCD camera. The experiments prove that although the actin filaments show an only-partial propensity for attachment in myosin-rich areas, their motility is confined to a large extent in micro-channels.

The affinity of chick cofilin for actin increases when actin is complexed with DNase I: formation of a cofilin-actin-DNase I ternary complex.

Cofilin, an actin‐binding protein, regulates the rate, nature and extent of assembly of the actin cytoskeleton. Native Phast gels show that the addition of cofilin to an actin‐DNase I complex (74 kDa) results in the formation of a ternary complex of 94 kDa indicating an equimolar stoichiometry in the ternary complex. Furthermore, native gels show that the addition of cofilin to a solution containing free actin and actin‐DNase I and run at pH 8.3 results in cofilin complexing preferentially to the actin‐DNase I complex. Conversely, the addition of DNase I to a solution containing an actin‐cofilin complex and free actin results in the preferential binding of DNase I to the actin‐cofilin complex. These results show that the affinity of cofilin for actin can be increased when actin forms binary complexes. When native gels were run at pH 6.8 the affinity of cofilin for monomeric actin was greater than for the actin‐DNase I complex indicating that the cofilin‐actin interaction can be regulated by changes in pH. The addition of cofilin to actin resulted in the polymerisation of actin at pH 6.8 whereas at alkaline pH a stable cofilin‐actin binary complex could be formed. The biological implications are discussed.

Micropatterning of Myosin on O-Acryloyl Acetophenone Oxime (AAPO), Layered with Bovine Serum Albumin (BSA)

This paper describes a simple laser-based method for preparing microchannels in a bilayer system consisting of a UV sensitive polymer, O-acryloyl acetophenone oxime (AAPO), layered with a protein-blocking agent, bovine serum albumin (BSA). Patterned surfaces suitable for biomolecular attachment are achieved through the use of a computer-controled laser ablation system, comprising a research-grade inverted optical microscope, a pulsed nitrogen laser emitting at 337 nm and a programmable X-Y-Z stage. Exposed areas with diameters of 5–20 μm, 1–5 μm, and sub-micron widths are readily achieved by focussing through a 20×dry objective, a 40×dry objective, or a 100×oil immersion lens, respectively. When combined with a sub-micron resolution, high-speed, computer-controlled X-Y-Z stage, well-defined channels or arrays can be patterned in the AAPO, revealing either the underlying hydrophobic primed-glass surface, or pendant amino groups suitable for the covalent binding of biomolecules, depending on the amount of energy delivered to the surface. The subsequent removal of the attached BSA creates well-defined regions with high contrast. Myosin was physically adsorbed to the base of the channels, and fluorescently-labeled actin microfilaments were observed to selectively bind to the myosin following ATP hydrolysis, confirming retention of bioactivity.

Electrophoretic monitoring of pollutants: effect of cations and organic compounds on protein interactions monitored by native gel electrophoresis.

We describe how the interaction between actin and its protein ligands can be used to evaluate the presence of certain metal (Cd, Cu, Hg, Zn) ions and organic compounds (2,4‐dioxin or Picloram) which are common components of environmental pollution. The assay detects the high‐affinity binding of actin to actin‐binding proteins (ABPs), cofilin or DNase I. The actin‐ABP complex was analyzed using native polyacrylamide gel electrophoresis and quantified by scanning densitometry. These proteins are widely distributed in animals and plant cells. The assay involves allowing the proteins to form an actin‐ABP complex into which increasing amounts of pollutants are titrated. Thus, the assay directly tests for inhibition of protein‐protein interaction. It is sensitive to common pollutants using concentration ranges over which they are known to exert a biological toxicity. A convenient feature of the assay is the fact that all the proteins can be stored in freeze‐dried form, and can be purchased commercially. We suggest that if this molecular assay is sensitive to a wide range of environmental pollutants, it could be used as a rapid and convenient assay of the environment in combination with currently available tests.

Regulation of the Cytoskeleton Assembly: a Role for a Ternary Complex of Actin with Two Actin-Binding Proteins

The actin cytoskeleton is an essential component of all eukaryotic cells. Besides its role in cell motility, it has a number of other functions, including cytokinesis, signal transduction, and the maintenance of cell shape. An essential property of the cytoskeleton is its ability to rapidly assemble and disassemble monomers of actin into F-actin filaments and this process is now known to be regulated by a number of actin-binding proteins (ABPs) of which cofilin appears to be the most widely distributed in nature. In this chapter we pose the question: does cofilin act alone in controlling actin filament assembly or is the binding of cofilin to actin modulated by other ABPs?

Surface Hydrophobicity Modulates the Operation of Protein Molecular Motors

The modulation of protein molecular motors (actin-myosin) motility has been tested on several candidate materials for microfluidics devices, all having different hydrophobicities, chemistries and nanotopographies. The analysis of the distribution of molecular properties on the molecular surface of the molecular motor protein suggests that the two very different, temporally separated, conformations of the heads exacerbate the impact of the adsorbing surface on protein behavior. The motility on surfaces with moderate hydrophobicity exhibits a bimodal distribution of velocities of actin filaments, which can be explained by the existence of two molecular conformations of surface-immobilized motor, i.e. with one or two free active heads that propel the actin filament. The study demonstrates that PMMA and not nitrocellulose - the classical choice for actin-myosin motility assays - is the optimum material for the fabrication of future nanofluidics devices based on protein molecular motors

A novel biosensor for mercuric ions based on motor proteins.

We explored the potential for use of the contractile proteins, actin and myosin, as biosensors of solutions containing mercury ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 μm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.

Controlling actin motility on microfabricated linear channels

Heavy meromyosin (HMM), a proteolytically cleaved derivative of myosin has previously been shown to interact with actin in well-established in vitro motility assays on nitrocellulose surfaces. In this study, the assays were conducted to demonstrate that the motility of actin filaments is confined in the micron-sized channels fabricated via laser ablation in a layer of the photosensitive resist polymer (O-acryloyloxime acetophenone oxime, AAPO). A solution containing myosin labelled with fluorophore 5-iodoacetamidofluorescein (5-IAF) was applied to the microfabricated AAPO surface and shown to bind specifically to the micron-size channels. In the motility assay, HMM, rhodamine-phalloidin labelled actin and ATP were sequentially added and the movement of the actin filaments was observed by fluorescence microscopy and recorded with a CCD camera. The experiments prove that although the actin filaments show an only-partial propensity for attachment in myosin-rich areas, their motility is confined to a large extent in micro- channels.