DNA Nunchucks: Nanoinstrumentation for Single-Molecule Measurement of Stiffness and Bending.

Bending of double-stranded DNA (dsDNA) has important applications in biology and engineering, but measurement of DNA bend angles is notoriously difficult and rarely dynamic. Here we introduce a nanoscale instrument that makes dynamic measurement of the bend in short dsDNAs easy enough to be routine. The instrument works by embedding the ends of a dsDNA in stiff, fluorescently labeled DNA nanotubes, thereby mechanically magnifying their orientations. The DNA nanotubes are readily confined to a plane and imaged while freely diffusing. Single-molecule bend angles are rapidly and reliably extracted from the images by a neural network. We find that angular variance across a population increases with dsDNA length, as predicted by the worm-like chain model, although individual distributions can differ significantly from one another. For dsDNAs with phased A6-tracts, we measure an intrinsic bend of 17 ± 1° per A6-tract, consistent with other methods, and a length-dependent angular variance that indicates A6-tracts are (80 ± 30)% stiffer than generic dsDNA.

Collective and single cell behavior in epithelial contact inhibition.

Control of cell proliferation is a fundamental aspect of tissue physiology central to morphogenesis, wound healing, and cancer. Although many of the molecular genetic factors are now known, the system level regulation of growth is still poorly understood. A simple form of inhibition of cell proliferation is encountered in vitro in normally differentiating epithelial cell cultures and is known as "contact inhibition." The study presented here provides a quantitative characterization of contact inhibition dynamics on tissue-wide and single cell levels. Using long-term tracking of cultured Madin-Darby canine kidney cells we demonstrate that inhibition of cell division in a confluent monolayer follows inhibition of cell motility and sets in when mechanical constraint on local expansion causes divisions to reduce cell area. We quantify cell motility and cell cycle statistics in the low density confluent regime and their change across the transition to epithelial morphology which occurs with increasing cell density. We then study the dynamics of cell area distribution arising through reductive division, determine the average mitotic rate as a function of cell size, and demonstrate that complete arrest of mitosis occurs when cell area falls below a critical value. We also present a simple computational model of growth mechanics which captures all aspects of the observed behavior. Our measurements and analysis show that contact inhibition is a consequence of mechanical interaction and constraint rather than interfacial contact alone, and define quantitative phenotypes that can guide future studies of molecular mechanisms underlying contact inhibition.

Distinct conformations of DNA-stabilized fluorescent silver nanoclusters revealed by electrophoretic mobility and diffusivity measurements.

Silver-DNA nanoclusters (Ag:DNAs) are novel fluorophores under active research and development as alternative biomolecular markers. Comprised of a few-atom Ag cluster that is stabilized in water by binding to a strand of DNA, they are also interesting for fundamental explorations into the properties of metal molecules. Here, we use in situ calibrated electrokinetic microfluidics and fluorescence correlation spectroscopy to determine the size, charge, and conformation of a select set of Ag:DNAs. Among them is a pair of spectrally distinct Ag:DNAs stabilized by the same DNA sequence, for which it is known that the silver cluster differs by two atoms. We find these two Ag:DNAs differ in size by ∼30%, even though their molecular weights differ by less than 3%. Thus a single DNA sequence can adopt very different conformations when binding slightly different Ag clusters. By comparing spectrally identical Ag:DNAs that differ in sequence, we show that the more compact conformation is insensitive to the native DNA secondary structure. These results demonstrate electrokinetic microfluidics as a practical tool for characterizing Ag:DNA.

Bilayer edges catalyze supported lipid bilayer formation.

Supported lipid bilayers (SLB) are important for the study of membrane-based phenomena and as coatings for biosensors. Nevertheless, there is a fundamental lack of understanding of the process by which they form from vesicles in solution. We report insights into the mechanism of SLB formation by vesicle adsorption using temperature-controlled time-resolved fluorescence microscopy at low vesicle concentrations. First, lipid accumulates on the surface at a constant rate up to approximately 0.8 of SLB coverage. Then, as patches of SLB nucleate and spread, the rate of accumulation increases. At a coverage of approximately 1.5 x SLB, excess vesicles desorb as SLB patches rapidly coalesce into a continuous SLB. Variable surface fluorescence immediately before SLB patch formation argues against the existence of a critical vesicle density necessary for rupture. The accelerating rate of accumulation and the widespread, abrupt loss of vesicles coincide with the emergence and disappearance of patch edges. We conclude that SLB edges enhance vesicle adhesion to the surface and induce vesicle rupture, thus playing a key role in the formation of continuous SLB.

Tau induces cooperative Taxol binding to microtubules.

Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds beta tubulin in the MT interior. Tau is a MT-associated protein that binds both alpha and beta tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of approximately 2 microM, and determines the escape rate of Taxol through one pore to be 1.7 x 10(3) (M x s)(-1). Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.

Variability-based sequence alignment identifies residues responsible for functional differences in alpha and beta tubulin.

alpha and beta Tubulin are well-characterized paralogs with similar structures and functions. We quantify the variability of every amino acid position in both tubulins from the aligned sequences of their numerous known orthologs. By aligning the variability profiles, we identify residues that differ significantly in variability between alpha and beta tubulin. Most of these residues are part of well-defined secondary structures and are clustered around the nucleotide binding pocket, the site of greatest functional difference between the two paralogs. The remaining residues of large difference in variability are located in the N-terminal loop between H1 and S2. We therefore predict that certain residues in this unstructured region also contribute to a functional difference between alpha and beta tubulin. Furthermore, we find the most restrictive variability-based alignment is nearly identical to the true structure-based alignment. Thus, by using a stringent variability-based alignment to approximate the true alignment, the method introduced here may predict sites of functional distinction between paralogous proteins even in the absence of structural information.

Mobility of taxol in microtubule bundles.

Mobility of taxol inside microtubules was investigated using fluorescence recovery after photobleaching on flow-aligned bundles. Bundles were made of microtubules with either GMPCPP or GTP at the exchangeable site on the tubulin dimer. Recovery times were sensitive to bundle thickness and packing, indicating that taxol molecules are able to move laterally through the bundle. The density of open binding sites along a microtubule was varied by controlling the concentration of taxol in solution for GMPCPP samples. With >63% sites occupied, recovery times were independent of taxol concentration and, therefore, inversely proportional to the microscopic dissociation rate, k(off). It was found that 10k(off)(GMPCPP) approximately equal k(off)(GTP), consistent with, but not fully accounting for, the difference in equilibrium constants for taxol on GMPCPP and GTP microtubules. With <63% sites occupied, recovery times decreased as approximately [Tax](-1/5) for both types of microtubules. We conclude that the diffusion of taxol inside the microtubule bundle is hindered by rebinding events when open sites are within approximately 7 nm of each other.

Size exclusion and diffusion of fluoresceinated probes within collagen fibrils.

The diffusion of fluoresceinated probes inside single collagen fibrils was investigated by imaging the migration of fluorescence along the fibrils in oil and by monitoring fluorescence recovery after photobleaching (FRAP). Probes were excluded from the fibrils according to their size. Probes that were not excluded diffused in the fibrils, but FRAP occurred 6 x 10(-4) times more slowly than in water due to binding interactions between collagen and the probes. The dissociation constant of the fluorescein-collagen complex was determined (K(D)=1.8+/-0.1 microM).

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay.

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.