Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB–DNA complexes has suggested that the enzyme promotes long-range DNA–DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg(2+), Ca(2+) or Mn(2+), suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge

Accurate determination of elastic parameters for multi-component membranes

Heterogeneities in the cell membrane due to coexisting lipid phases have been conjectured to play a major functional role in cell signaling and membrane trafficking. Thereby the material properties of multiphase systems, such as the line tension and the bending moduli, are crucially involved in the kinetics and the asymptotic behavior of phase separation. In this Letter we present a combined analytical and experimental approach to determine the properties of phase-separated vesicle systems. First we develop an analytical model for the vesicle shape of weakly budded biphasic vesicles. Subsequently experimental data on vesicle shape and membrane fluctuations are taken and compared to the model. The combined approach allows for a reproducible and reliable determination of the physical parameters of complex vesicle systems. The parameters obtained set limits for the size and stability of nanodomains in the plasma membrane of living cells.Comment: (*) authors contributed equally, 6 pages, 3 figures, 1 table; added insets to figure

Sarcomeric Pattern Formation by Actin Cluster Coalescence

Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Nucleic Acid Templated Chemical Reaction in a Live Vertebrate

Nucleic acid templated reactions are enabled by the hybridization of probe-reagent conjugates resulting in high effective reagent concentration and fast chemical transformation. We have developed a reaction that harnesses cellular microRNA (miRNA) to yield the cleavage of a linker releasing fluorogenic rhodamine in a live vertebrate. The reaction is based on the catalytic photoreduction of an azide by a ruthenium complex. We showed that this system reports specific expression of miRNA in living tissues of a vertebrate

Quantification of Biological Interactions with Particle Image Cross-Correlation Spectroscopy (PICCS)

A multitude of biological processes that involve multiple interaction partners are observed by two-color microscopy. Here we describe an analysis method for the robust quantification of correlation between signals in different color channels: particle image cross-correlation spectroscopy (PICCS). The method, which exploits the superior positional accuracy obtained in single-object and single-molecule microscopy, can extract the correlation fraction and length scale. We applied PICCS to correlation measurements in living tissues. The morphogen Decapentaplegic (Dpp) was imaged in wing imaginal disks of fruit fly larvae and we quantified what fraction of early endosomes contained Dpp