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Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations Precisely measuring the ensemble of conformers that a macromolecule populates in solution is highly challenging. Thus, it has been difficult to confirm or falsify the predictions of nanometer-scale dynamical modeling. Here, we apply an X-ray interferometry technique to probe the solution structure and fluctuations of B-form DNA on a length scale comparable to a protein-binding site. We determine an extensive set of intrahelix distance distributions between pairs of probes placed at distinct points on the surface of the DNA duplex. The distributions of measured distances reveal the nature and extent of the thermally driven mechanical deformations of the helix. We describe these deformations in terms of elastic constants, as is common for DNA and other polymers. The average solution structure and microscopic elasticity measured by X-ray interferometry are in striking agreement with values derived from DNA–protein crystal structures and measured by force spectroscopy, with one exception. The observed microscopic torsional rigidity of DNA is much lower than is measured by single-molecule twisting experiments, suggesting that torsional rigidity increases when DNA is stretched. Looking forward, molecular-level interferometry can provide a general tool for characterizing solution-phase structural ensembles.
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Genomic responses in mouse models poorly mimic human inflammatory diseases A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R2 between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases. PRESS COVERAGE
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Radial Construction of an Arterial Wall Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding
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A cell-free system for functional centromere and kinetochore assembly This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.
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Imaging nanometre-scale structure in cells using in situ aberration correction Accurate distance measurements of cellular structures on a length scale relevant to single macromolecules or macromolecular complexes present a major challenge for biological microscopy. In addition to the inherent challenges of overcoming the limits imposed by the diffraction of light, cells themselves are a complex and poorly understood optical environment. We present an extension of the high-resolution colocalization method to measure three dimensional distances between diffraction-limited objects using standard widefield fluorescence microscopy. We use this method to demonstrate that in three dimensions, cells intrinsically introduce a large and variable amount of chromatic aberration into optical measurements. We present a means of correcting this aberration in situ [termed ‘Colocalization and In-situ Correction of Aberration for Distance Analysis’ (CICADA)] by exploiting the fact that there is a linear relationship between the degree of aberration between different wavelengths. By labelling a cellular structure with redundantly multi-colour labelled antibodies, we can create an intracellular fiducial marker for correcting the individual aberrations between two different wavelengths in the same cells. Our observations demonstrate that with suitable corrections, nanometre scale three-dimensional distance measurements can be used to probe the substructure of macromolecular complexes within cells. |
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The split personality of CENP-A nucleosomes A specialized chromatin domain, called the centromere, ensures accurate chromosome segregation during mitosis. Centromeres are the foundation for the assembly of the kinetochore, the site on each chromosome that acts as the primary interface between the chromosomes and the microtubules of the mitotic spindle. Maintaining the centromere is therefore essential for chromosome stability. The chromatin mark that determines centromere identity is a specialized histone H3 variant called CENP-A (called Cse4 in the budding yeast Saccharomyces cerevisiae). Budding yeast assemble Cse4 into chromatin during each round of DNA replication, whereas vertebrate CENP-A nucleosome assembly is replication independent, occurring during telophase and G1. |
Publications Archive PubMed Site |
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