Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca2+-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the a-actinin/ 3-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to 0/y-actin from bovine spleen but not to a-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of proteinprotein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observed in vitro at an antibody concentration of 1.2 g/ml over 12 h. The effect of TRL1068 in vivo was evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections.T he understanding of bacterial physiology has fundamentally changed since the discovery of biofilms in the bacterial life cycle (1-3). Biofilms provide an anchor and physical protection for bacterial cells and the physiology and genetic programming of the bacteria shift from the planktonic (free-floating) to a sessile (adherent) state. This shift can result in a substantial reduction of antibiotic sensitivity in the biofilm (4). As much as 65 to 80% of clinically significant bacterial infections resistant to antibiotics are associated with biofilm (5, 6), including those of implants and catheters, infective endocarditis, lung infections associated with cystic fibrosis and chronic obstructive pulmonary disease (COPD), persistent infections of the ears and urinary tract, osteomyelitis, and surgery-associated nosocomial infections. Accordingly, a promising approach to treatment is to disrupt biofilms so that the freed bacteria become sensitive to available antibiotics as well as more fully subject to immune control (7).Biofilms are not simply random assemblies of bacterial and host components. Rather, the polymers in a biofilm form a multinode scaffolding with a semirigid, three-dimensional web-like architecture (8) which serves to exclude host immune cells while allowing the diffusion of nutrients and waste. Comparative genomic studies have identified tens of proteins a...

Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.