DataSheet1.pdf

<p>Cell division in most prokaryotes is mediated by the well-studied fts genes, with FtsZ as the principal player. In many archaeal species, however, division is orchestrated differently. The Crenarchaeota phylum of archaea features the action of the three proteins, CdvABC. This Cdv system is a unique and less-well-studied division mechanism that merits closer inspection. In vivo, the three Cdv proteins form a composite band that contracts concomitantly with the septum formation. Of the three Cdv proteins, CdvA is the first to be recruited to the division site, while CdvB and CdvC are thought to participate in the active part of the Cdv division machinery. Interestingly, CdvB shares homology with a family of proteins from the eukaryotic ESCRT-III complex, and CdvC is a homolog of the eukaryotic Vps4 complex. These two eukaryotic complexes are key factors in the endosomal sorting complex required for transport (ESCRT) pathway, which is responsible for various budding processes in eukaryotic cells and which participates in the final stages of division in Metazoa. There, ESCRT-III forms a contractile machinery that actively cuts the membrane, whereas Vps4, which is an ATPase, is necessary for the turnover of the ESCRT membrane-abscission polymers. In contrast to CdvB and CdvC, CdvA is unique to the archaeal Crenarchaeota and Thaumarchaeota phyla. The Crenarchaeota division mechanism has often been suggested to represent a simplified version of the ESCRT division machinery thus providing a model system to study the evolution and mechanism of cell division in higher organisms. However, there are still many open questions regarding this parallelism and the division mechanism of Crenarchaeota. Here, we review the existing data on the role of the Cdv proteins in the division process of Crenarchaeota as well as concisely review the ESCRT system in eukaryotes. We survey the similarities and differences between the division and abscission mechanisms in the two cases. We suggest that the Cdv system functions differently in archaea than ESCRT does in eukaryotes, and that, unlike the eukaryotic case, the Cdv system's main function may be related to surplus membrane invagination and cell-wall synthesis.</p

Detection of Nucleosomal Substructures using Solid-State Nanopores

Histone proteins assemble onto DNA into nucleosomes that control the structure and function of eukaryotic chromatin. More specifically, the structural integrity of nucleosomes regulates gene expression rates and serves as an important early marker for cell apoptosis. Nucleosomal (sub)­structures are however hard to detect and characterize. Here, we show that solid-state nanopores are well suited for fast and label-free detection of nucleosomes and its histone subcomplexes. (Nucleo-)­protein complexes are individually driven through the nanopore by an applied electric field, which results in characteristic conductance blockades that provide quantitative information on the molecular size of the translocating complex. We observe a systematic dependence of the conductance blockade and translocation time on the molecular weight of the nucleosomal substructures. This allows discriminating and characterizing these protein and DNA–protein complexes at the single-complex level. Finally, we demonstrate the ability to distinguish nucleosomes and dinucleosomes as a first step toward using the nanopore platform to study chromatin arrays

Measurement of the Docking Time of a DNA Molecule onto a Solid-State Nanopore

We present measurements of the change in ionic conductance due to double-stranded (ds) DNA translocation through small (6 nm diameter) nanopores at low salt (100 mM KCl). At both low (<200 mV) and high (>600 mV) voltages we observe a current enhancement during DNA translocation, similar to earlier reports. Intriguingly, however, in the intermediate voltage range, we observe a new type of composite events, where within each single event the current first decreases and then increases. From the voltage dependence of the magnitude and timing of these current changes, we conclude that the current decrease is caused by the docking of the DNA random coil onto the nanopore. Unexpectedly, we find that the docking time is exponentially dependent on voltage (<i>t</i> ∝ e^{–<i>V</i>/<i>V</i>₀}). We discuss a physical picture where the docking time is set by the time that a DNA end needs to move from a random location within the DNA coil to the nanopore. Upon entrance of the pore, the current subsequently increases due to enhanced flow of counterions along the DNA. Interestingly, these composite events thus allow to independently measure the actual translocation time as well as the docking time before translocation

Plasmonic Nanopore for Electrical Profiling of Optical Intensity Landscapes

We present a novel method for sensitive mapping of optical intensity distributions at subdiffraction-limited resolution. This is achieved with a novel device, a plasmonic nanopore, which combines a plasmonic bowtie nanoantenna with a 10 nm-in-diameter solid-state nanopore. Variations in the local optical intensity modulate the plasmonic heating, which we measure electrically through changes in the ionic conductance of the nanopore. We demonstrate the method by profiling the focal volume of a 10 mW laser beam that is tightly focused by a high-numerical-aperture microscope objective. The results show a complex three-dimensional intensity distribution that closely matches predictions obtained by theoretical calculations of the optical system. In addition to laser profiling, the ionic conductance of a nanopore is also shown to provide quantitative estimates of the temperature in the proximity of single plasmonic nanostructures

DNA extension as a function of supercoiling density.

<p>For six DNA sequences, the DNA extension <i>Z</i> is measured as function of the applied supercoiling density at varying forces between 0.2 and 1.2 pN. The shown curves are averages of a large number of individual molecules (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#sec002" target="_blank">Materials and Methods</a>). At 0.3pN and below all rotation curves are symmetric and applied supercoiling is absorbed into plectoneme formation. Increasing the applied stretching force results in less symmetric rotation curves. At intermediate forces of 0.6 and 0.8pN the sequence-dependent effect of competition between DNA melting and plectoneme formation is most pronounced. At 1.2pN all sequences show an asymmetric rotation curve: applied negative supercoiling is absorbed into DNA melting, and applied positive supercoiling is absorbed into plectonemes. For ease of comparison with other work, on the right axis the extension is presented relative to the contour length of the molecule (3.33μm). The noise in the extension (<i>std</i> of <i>Z</i>) of the molecules for these curves are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#pone.0141576.s002" target="_blank">S2 Fig</a>.</p

Difference in extension and noise at <i>σ</i> = ±0.05.

<p>(A) The difference in extension <i>ΔZ</i> = 〈<i>Z</i>(<i>σ</i> = −0.05)〉 − 〈<i>Z</i>(<i>σ</i> = +0.05)〉, as a function of applied stretching force. An increase in GC content results in a larger <i>ΔZ</i>, indicating more melting, which occurs at a lower applied stretching force. To determine the characteristic melting force <i>F</i>_<i>char</i>, the force at half of the maximum <i>ΔZ</i> value per sequence is determined (open circles). The extension <i>Z</i> as function of force and <i>ΔZ</i> at <i>σ</i> = ± 0.02, 0.03, 0.04, 0.05 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#pone.0141576.s004" target="_blank">S4A Fig</a>. On the right axis the extension is presented relative to the contour length of the molecule (3.33μm). (B) To quantify the noise on the signal in <i>Z</i>, the standard deviation in <i>Z</i> was measured for each molecule at each force and supercoiling density individually. For all molecules with the same sequence, the standard deviations under the same condition are averaged. This figure shows the force dependence of the difference in standard deviation at <i>σ</i> ± 0.05, Δ<i>std</i> = 〈<i>std</i>(<i>σ</i> = −0.05)〉 − 〈<i>std</i>(<i>σ</i> = +0.05)〉. A large standard deviation in <i>Z</i> at negative <i>σ</i> compared to positive <i>σ</i> in the force regime around 0.6–0.8 pN is indicative of the denaturation transition. To find the force at which the maximum in <i>∆std</i> occurs, a Gaussian fit to <i>∆std</i> has been made. The maximum in <i>∆std</i> (marked by stars) occurs at lower forces for GC-rich sequences, in agreement with the increase in <i>ΔZ</i> at lower forces for GC-rich sequences as shown in Fig A). More data on the <i>std</i> and <i>∆std</i> at different supercoiling densities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#pone.0141576.s004" target="_blank">S4B Fig</a>. (C) The characteristic force <i>F</i>_<i>char</i> as function of the GC content. The open circles are the <i>F</i>_<i>char</i> values as determined from the <i>ΔZ</i> data. The stars are the <i>F</i>_<i>char</i> values as determined from the <i>Δstd</i> data for |<i>σ</i>| = 0.04, and 0.05. Similar values for <i>F</i>_<i>char</i> are obtained from <i>ΔZ</i> and <i>Δstd</i>. The black solid line is a phenomenological fit of <i>F</i>_<i>char</i> = (11/(%GC) + 0.4) pN. The transition from plectonemic to melted DNA occurs at lower applied stretching force for GC-rich sequences.</p

Length increase due to melting.

<p>The length increase due to melting, <i>ΔZ</i> is calculated from the extension at negative supercoiling density minus the extension at similar positive supercoiling density, <i>ΔZ</i> = 〈<i>Z</i>(−|<i>σ</i>|)〉 − 〈<i>Z</i>(+|<i>σ</i>|)〉. (A) Illustration of <i>ΔZ</i> with the data of the 77%GC construct at 1.0pN. (B-F) <i>ΔZ</i> as function of supercoiling density <i>σ</i> for forces between 0.5pN and 1.2pN. A positive <i>ΔZ</i> occurs when applied negative supercoiling induces melting where similar positive supercoiling induces plectonemes. At forces below 0.5pN, <i>ΔZ</i> is zero and no melting occurs. An increase in GC content results in a larger amount of melting and a smaller amount of plectoneme formation at the same force. The dotted lines at <i>σ</i> = -0.05 indicate the values of <i>ΔZ</i> used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#pone.0141576.g004" target="_blank">Fig 4A</a> in which the force dependence of <i>ΔZ</i> is shown. On the right axis the extension is presented relative to the contour length of the molecule (3.33μm). The difference in noise between positive and negative supercoiling (<i>∆std</i>) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141576#pone.0141576.s003" target="_blank">S3 Fig</a>.</p

A Simple Self-Calibrating Method To Measure the Height of Fluorescent Molecules and Beads at Nanoscale Resolution

We describe a simple self-calibrating technique, incident-beam interference sweeping, for measuring the height of fluorescent labels. Using a tilted back-reflecting mirror and a scanning laser beam, a modulated fluorescence emission allows height determination of a label from a surface with a resolution of ∼3 nm. In addition, we show that the absolute distance of a label from the top-mounted mirror can be determined with a resolution of a few tens of nanometers over a micrometer range

Improvement in measuring axial position using skew position shift rather than mean.

<p>Top row) Left) The calculated extension from simulations through using either the mean (blue diamonds) or the skew distribution position (red circles) as a function of nominal extension expected from the WLC, for a tether. The black line indicates measurement equal to the nominal extension. Right) Residuals squared for difference between measured and nominal extension using same data as left. Bottom row) Same as top except for tether. Error bars are standard error of the mean with n = 5.</p

The difference in end-to-end length of the peak of the rotation curves before and after the assembly experiment, plotted against the change in supercoiling state as deduced from the shift of the maximum of the rotation curve (ΔL_k,nuc).

<p>Black squares show the results for experiments of NAP1 preincubated with all four histones assembled at 0.3 pN, grey triangles at 1 pN. Blue stars are the results from experiment of NAP1 preincubated with histones H3 and H4 only. A linear fit through the origin of the black and grey data (red line) reveals a slope of 56±3 nm per turn (red line). If one assumes that the assembly of one nucleosome results in the formation of 1 positive supercoil, this means that the assembly of one nucleosome decreases the end-to-end length by 56 nm at 0.3 pN. For comparison, lines with a slope of 40 (black) and 80 (green) nm decrease per negative unit change in linking number ΔL_k,nuc are shown. The experiments with H3 and H4 only do show a decrease in end-to-end length but do not show a change in linking number.</p