Min proteins of the Escherichia coli cell division system oscillate between the cell poles in vivo. In vitro on a solid-surface supported lipid bilayer, these proteins exhibit a number of interconverting modes of collective ATP-driven dynamic pattern formation including not only the previously described propagating waves, but also near uniformity in space surface concentration oscillation, propagating filament like structures with a leading head and decaying tail and moving and dividing amoeba-like structures with sharp edges. We demonstrate that the last behavior most closely resembles in vivo system behavior. The simple reaction-diffusion models previously proposed for the Min system fail to explain the results of the in vitro self-organization experiments. We propose the hypotheses that initiation of MinD binding to the surface is controlled by counteraction of initiation and dissociation complexes; the binding of MinD/E is stimulated by MinE and involves polymerization-depolymerization dynamics; polymerization of MinE over MinD oligomers triggers dynamic instability leading to detachment from the membrane. The physical properties of the lipid bilayer are likely to be one of the critical determinants of certain aspects of the dynamic patterns observed.cell division | min system | oscillations and waves | self-organization | septum localization

We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe. W e describe measurements of nanometer-scale conformational changes of single DNA oligonucleotides, 40-90 bases long. The experiments are based on a micromechanical technique, which we have introduced previously (1). By measuring the contour length shortening of a single-probe molecule on formation of the double-stranded (ds) structure, we apply the method to detect hybridization of single unlabeled target oligomers. There are two interesting aspects to this approach. First, the ability to monitor directly certain conformational changes of DNA and RNA provides a tool for investigating processes such as (protein-induced) bending and looping, which have regulatory roles in transcription and splicing. The most detailed information on static conformations is provided by labor-intensive structural studies. At the other end, simple gel-shift assays provide a partial characterization, such as a bending angle. Single-molecule methods, in principle, offer a direct way of studying conformational changes, including dynamics. Recently, the conformational change involved in the catalytic activity of a ribozyme has been studied in detail by single-molecule fluorescence resonance energy transfer (FRET) (2). However, FRET is limited to distances Ͻ10 nm, whereas many interesting structures formed by DNA, such as bends and loops, involve larger (10-to 30-nm) scales (10 nm is the contour length of a dsDNA 30 mer). With the method described here, we can detect nanometer-scale conformational changes of single DNA oligomers of length 30-90 bases, thus extending the range covered by FRET.A second aspect is the possibility of developing sensitive assays based on single-molecule detection. DNA hybridization assays are ubiquitous in genomic analysis, gene expression studies, and, increasingly, diagnostics. The sensitivity and throughput of the assays have recently been improved through the introduction of DNA arrays and the development of several new sensitive detection techniques. These include molecular beacons (3-6), nanoparticle composites (7-10), surface plasmon resonance (11, 12), fiber-optic arrays (13-16), and conductivity͞capacitance measurements (17, 18). The most widely used detection methods rely on labeling target DNA, usually by fluorescent dyes. However, the resulting sensitivity limits the range of applications, specifically for DNA arrays where small populations of cells are to be analyzed. Thus improved sensitivity would be valuable.With...

Background: Endosomal sorting complex required for transport (ESCRT) complexes sort ubiquitinated membrane proteins into vesicles that bud away from the cytosol. Results: Human ESCRT-II assembles into cholesterol-dependent clusters of 10 -100 molecules that bind ubiquitin and the ESCRT-III subunit VPS20 and exclude an L d -phase-specific dye. Conclusion: ESCRT proteins induce lipid phase separation in vitro. Significance: Lipid phase separation by ESCRTs may aid membrane budding.

We propose a statistical mechanics model for DNA melting in which base stacking and pairing are explicitly introduced as distinct degrees of freedom. Unlike previous approaches, this model describes thermal denaturation of DNA secondary structure in the whole experimentally accessible temperature range. Base pairing is described through a zipper model, base stacking through an Ising model. We present experimental data on the unstacking transition, obtained exploiting the observation that at moderately low pH this transition is moved down to experimentally accessible temperatures. These measurements confirm that the Ising model approach is indeed a good description of base stacking. On the other hand, comparison with the experiments points to the limitations of the simple zipper model description of base pairing.