Bulletin of the American Physical Society
APS March Meeting 2014
Volume 59, Number 1
Monday–Friday, March 3–7, 2014; Denver, Colorado
Session F15: Focus Session: Physics of Chromosomes |
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Sponsoring Units: DBIO Chair: Dieter Heerman, University of Heidelberg Room: 304 |
Tuesday, March 4, 2014 8:00AM - 8:36AM |
F15.00001: Mechanics, Structure and Dynamics of Metaphase Chromosome Folding Invited Speaker: John F. Marko During cell division, eukaryote chromosomes are restructured from a relatively dispersed interphase form, into a relatively compact folded metaphase form. I will discuss experiments aimed at analyzing the folding scheme of metaphase chromosomes, where mechanical response and biochemical perturbation are used as tools for diagnosing structure. Experiments with nucleases reveal that the continuity of the metaphase chromosome depends on DNA, i.e., that the metaphase chromosome can be considered to be a ``chromatin gel.'' Experiments with topoisomerases indicate that chromatin entanglements play an appreciable role in determining chromosome mechanical properties, suggesting that they may play a structural role. We further show that perturbation of condensin complexes dramatically changes metaphase chromosome mechanics. Finally we report results of fluorescence visualization of distributions of condensin I and II along metaphase chromosomes. [Preview Abstract] |
Tuesday, March 4, 2014 8:36AM - 8:48AM |
F15.00002: Understanding DNA Condensation: From Simple Ions to Protamine-DNA Packaging in Sperm Jason DeRouchey DNA in nature exists primarily in a highly compacted state critical for most biological functions. DNA condensation, however, remains poorly understood at the molecular level. We are interested in understanding the fundamental interactions, molecular scale forces and elucidating mechanisms by which polycations interact with DNA in vitro and in vivo. We use osmotic stress coupled with x-ray scattering, to study packaging densities and compaction energies between DNA helices in the presence of various cations. In this talk, we will discuss from simple ions to complex proteins and how these cations modulate both the attractive and repulsive forces between DNA helices. Lastly, the biological implications of these forces will be discussed with regards to spermatogenesis where chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging from spermatogenesis is considered essential for both successful transport as well as to protect DNA from damage. [Preview Abstract] |
Tuesday, March 4, 2014 8:48AM - 9:00AM |
F15.00003: Folding of Nucleosome Arrays Steven Howell, Isabel Jimenez-Useche, Kurt Andresen, Chongli Yuan, Xiangyun Qiu Chromatin conformation and dynamics is central to gene functions including packaging, regulation, and repair. At the molecular level, the basic building block of chromatin is a nucleosome core particle (NCP) made of ~147 base pairs (bp) of dsDNA wrapped around an octamer of histone proteins. These NCPs are connected by short 10-90 bps of linker DNA as beads on a string. Key factors determining the packaging of NCP arrays to form chromatin include ionic condition, linker DNA length, and epigenetic modifications, especially of the histone tails. We have investigated how the conformations of model tetra-NCP arrays are modulated by these factors using small angle x-ray scattering (SAXS). Here we present recent studies of the effects of ion (KCl and MgCl2), linker length, and histone modification (tail deletions) on NCP arrays. Our SAXS measurement makes it possible to learn about both the global compaction of NCP arrays and local inter-NCP spatial correlations within the same array. [Preview Abstract] |
Tuesday, March 4, 2014 9:00AM - 9:12AM |
F15.00004: Entropy in DNA Double-Strand Break, Detection and Signaling Yang Zhang, Christina Schindler, Dieter Heermann In biology, the term entropy is often understood as a measure of disorder - a restrictive interpretation that can even be misleading. Recently it has become clearer and clearer that entropy, contrary to conventional wisdom, can help to order and guide biological processes in living cells. DNA double-strand breaks (DSBs) are among the most dangerous lesions and efficient damage detection and repair is essential for organism viability. However, what remains unknown is the precise mechanism of targeting the site of damage within billions of intact nucleotides and a crowded nuclear environment, a process which is often referred to as recruitment or signaling. Here we show that the change in entropy associated with inflicting a DSB facilitates the recruitment of damage sensor proteins. By means of computational modeling we found that higher mobility and local chromatin structure accelerate protein association at DSB ends. We compared the effect of different chromatin architectures on protein dynamics and concentrations in the vicinity of DSBs, and related these results to experiments on repair in heterochromatin. Our results demonstrate how entropy contributes to a more efficient damage detection. We identify entropy as the physical basis for DNA double-strand break signaling. [Preview Abstract] |
Tuesday, March 4, 2014 9:12AM - 9:24AM |
F15.00005: Nanofluidic laboratory-on-chip device for mapping of single molecule DNA extracted from single cells Sara Mahshid, Daniel Berard, Robert Sladek, Sabrina Leslie, Walter Reisner The aim of this project is to create a nanofluidic platform to provide comprehensive maps of single-cell genomes at 1 kbp resolution based on the direct analysis of single 1-10 Mbp extended DNA molecules extracted from individual cells on-chip. We have developed a nanodevice in which all biochemical processing of single cells (cell lysis, DNA purification and fragmentation) is performed in situ. The platform has the following three components: (1) a micro-cavity (50$\times$20 micron in dimension) for trapping and biochemical processing of single cells; (2) post arrays (1 micron depth) for untangling the released genomic contents and (3) parallel nanochannel arrays (100 nm) for extension of $\sim$ 1-10 Mbp DNA for high-throughput optical mapping. Moreover, we use ``Convex Lense-Induced Nanoconfinement'' (CLIC) technique for trapping of single cell and dragging DNA into nanochannels. The principle is that a convex lens is pushed down to deform a flexible coverslip lid above the aforesaid platform containing nano/micro patterns, creating a locally confined region that pins molecules in the embedded nano/micro features. CLIC is used to lower the device lid over a cell isolated in the microcavity with an adjustable gap for buffer exchange. The released DNA is untangled using 1 micron-deep post arrays and driven into nanochannel array where its genomic content is revealed. In particular, using CLIC we were able to successfully trap 20 micron lymphoblast cells inside microcavity and lyse the trapped cell to drive out DNA. [Preview Abstract] |
Tuesday, March 4, 2014 9:24AM - 9:36AM |
F15.00006: Active microrheology of a model of the nuclear micromechanical environment Henry Byrd, Maria Kilfoil In order to successfully complete the final stages of chromosome segregation, eukaryotic cells require the motor enzyme topoisomerase II, which can resolve topological constraints between entangled strands of duplex DNA. We created an in vitro model of a close approximation of the nuclear micromechanical environment in terms of DNA mass and entanglement density, and investigated the influence of this motor enzyme on the DNA mechanics. Topoisomerase II is a non-processive ATPase which we found significantly increases the motions of embedded microspheres in the DNA network. Because of this activity, we study the mechanical properties of our model system by active microrheology by optical trapping. We test the limits of fluctuation dissipation theorem (FDT) under this type of activity by comparing the active microrheology to passive measurements, where thermal motion alone drives the beads. We can relate any departure from FDT to the timescale of topoisomerase II activity in the DNA network. These experiments provide insight into the physical necessity of this motor enzyme in the cell. [Preview Abstract] |
Tuesday, March 4, 2014 9:36AM - 10:12AM |
F15.00007: Models of chromatin spatial organisation in the cell nucleus Invited Speaker: Mario Nicodemi In the cell nucleus chromosomes have a complex architecture serving vital functional purposes. Recent experiments have started unveiling the interaction map of DNA sites genome-wide, revealing different levels of organisation at different scales. The principles, though, which orchestrate such a complex 3D structure remain still mysterious. I will overview the scenario emerging from some classical polymer physics models of the general aspect of chromatin spatial organisation. The available experimental data, which can be rationalised in a single framework, support a picture where chromatin is a complex mixture of differently folded regions, self-organised across spatial scales according to basic physical mechanisms. I will also discuss applications to specific DNA loci, e.g. the HoxB locus, where models informed with biological details, and tested against targeted experiments, can help identifying the determinants of folding. [Preview Abstract] |
Tuesday, March 4, 2014 10:12AM - 10:24AM |
F15.00008: Is DNA a non-draining, swollen coil? Abhiram Muralidhar, Douglas Tree, Patrick Doyle, Kevin Dorfman Double-stranded DNA has long been used as a model polymer in a wide variety of experiments, particularly in single molecule studies. However, there is little consensus about whether molecules used commonly in experiments, such as $\lambda$-DNA (48.5 kbp, kilo base pairs) and T4-DNA (169 kbp), are long enough to exhibit universal, long-chain behavior. To resolve this point of contention, we use Pruned-Enriched Rosenbluth Method (PERM) simulations to calculate static and near-equilibrium dynamic properties of DNA ranging from a molecular weight of 100 bp to nearly 1 Mbp (mega base pairs). By evaluating metrics such as the end-to-end distance, and comparing these results with renormalization group theory predictions, we show that molecules such as $\lambda$-DNA and T4-DNA are far from the swollen coil limit. Our results indicate that DNA exhibits flexible swollen coil behavior when the contour length is approximately 1 Mbp. Moreover, computation of the Kirkwood diffusivity from equilibrium configurations reveals that DNA is partially draining to chain lengths as big as 1 Mbp. We attribute this slow transition to universal behavior to the semiflexible nature of DNA, that gives rise to weak intramolecular excluded volume and hydrodynamic interactions. [Preview Abstract] |
Tuesday, March 4, 2014 10:24AM - 10:36AM |
F15.00009: Escape of a knot from a DNA molecule in flow Benjamin Renner, Patrick Doyle Macroscale knots are an everyday occurrence when trying to unravel an unorganized flexible string (e.g. an iPhone cord taken out of your pocket). In nature, knots are found in proteins and viral capsid DNA, and the properties imbued by their topologies are thought to have biological significance. Unlike their macroscale counterparts, thermal fluctuations greatly influence the dynamics of polymer knots. Here, we use Brownian Dynamics simulations to study knot diffusion along a linear polymer chain. The model is parameterized to dsDNA, a model polymer used in previous simulation and experimental studies of knot dynamics. We have used this model to study the process of knot escape and transport along a dsDNA strand extended by an elongational flow. For a range of knot topologies and flow strengths, we show scalings that result in collapse of the data onto a master curve. We show a topologically mediated mode of transport coincides with observed differences in rates of knot transport, and we provide a simple mechanistic explanation for its effect. We anticipate these results will build on the growing body of fundamental studies of knotted polymers and inform future experimental study. [Preview Abstract] |
Tuesday, March 4, 2014 10:36AM - 10:48AM |
F15.00010: Depletion Zone Effects in Active Microrheology Studies of DNA Solutions Cole D. Chapman, Douglas E. Smith, Rae M. Robertson-Anderson In active microrheology studies, micron scale spheres are driven through complex fluids while forces imparted on the spheres are measured to determine valuable information about the fluids at the molecular level. However, when a microsphere is dragged through a polymer solution, polymers can amass along its leading surface while leaving an area devoid of polymers in its wake (depletion zone). Depletion zone effects can complicate the interpretation of the measured force, prohibiting standard continuum limit methods for analyzing microrheology data. Here, we examine the depletion zone created by dragging microspheres embedded within a network of DNA (a model polymer). Using dual-force optical tweezers, parallel microspheres are driven axially through a DNA solution, while measuring the force imparted on the individual microspheres. Thus, we are able to explore the effective `wake' created by the leading microsphere via the response of the lagging microsphere and its dependence on a variety of parameters, such as solution concentration, distance traveled, and driving rate. This technique is combined with single-molecule fluorescence microscopy, allowing for simultaneous visualization of the deformation of the individual DNA molecules surrounding the driven microspheres. [Preview Abstract] |
Tuesday, March 4, 2014 10:48AM - 11:00AM |
F15.00011: Dual-Colored DNA Comb Polymers for Single Molecule Rheology Danielle Mai, Amanda Marciel, Charles Schroeder We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions. [Preview Abstract] |
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