The layered organization of nucleosomes in 30 nm chromatin fibers

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10–11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5–6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.     

Stretching DNA with optical tweezers.

Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.     

Binding of TmHU to single dsDNA as observed by optical tweezers

Optical tweezers are employed to study the action of the histone-like protein from Thermotoga maritima (TmHU) on DNA at a single molecule level. Binding and disruption of TmHU to and from DNA are found to take place in discrete steps of 4-5 nm length and a net binding enthalpy of about 16kBT. This is in reasonable agreement with a microscopic model that estimates the extension of the binding sites of the protein and evaluates the energetics mainly for bending of the DNA in the course of interaction.     

Spectrin-level modeling of the cytoskeleton and optical tweezers stretching of the erythrocyte

We present a three-dimensional computational study of whole-cell equilibrium shape and deformation of human red blood cell (RBC) using spectrin-level energetics. Random network models consisting of degree-2, 3, ..., 9 junction complexes and spectrin links are used to populate spherical and biconcave surfaces and intermediate shapes, and coarse-grained molecular dynamics simulations are then performed with spectrin connectivities fixed. A sphere is first filled with cytosol and gradually deflated while preserving its total surface area, until cytosol volume consistent with the real RBC is reached. The equilibrium shape is determined through energy minimization by assuming that the spectrin tetramer links satisfy the worm-like chain free-energy model. Subsequently, direct stretching by optical tweezers of the initial equilibrium shape is simulated to extract the variation of axial and transverse diameters with the stretch force. At persistence length p = 7.5 nm for the spectrin tetramer molecule and corresponding in-plane shear modulus mu(0) approximately 8.3 microN/m, our models show reasonable agreement with recent experimental measurements on the large deformation of RBC with optical tweezers. We find that the choice of the reference state used for the in-plane elastic energy is critical for determining the equilibrium shape. If a position-independent material reference state such as a full sphere is used in defining the in-plane energy, then the bending modulus kappa needs to be at least a decade larger than the widely accepted value of 2 x 10(-19) J to stabilize the biconcave shape against the cup shape. We demonstrate through detailed computations that this paradox can be avoided by invoking the physical hypothesis that the spectrin network undergoes constant remodeling to always relax the in-plane shear elastic energy to zero at any macroscopic shape, at some slow characteristic timescale. We have devised and implemented a liquefied network structure evolution algorithm that relaxes shear stress everywhere in the network and generates cytoskeleton structures that mimic experimental observations.     

Detection and characterization of individual intermolecular bonds using optical tweezers

The development of scanning probe techniques has made it possible to examine protein-protein interactions at the level of individual molecular pairs. A calibrated optical tweezers, along with immunoglobulin G (IgG)-coated polystyrene microspheres, has been used to detect individual surface-linked Staphylococcus protein A (SpA) molecules and to characterize the strength of the noncovalent IgG-SpA bond. Microspheres containing, on average, less than one IgG per contact area were held in the optical trap while an SpA-coated substrate was scanned beneath them at a distance of approximately 50 nm. This geometry allows the trapped bead to make contact with the surface, from bond formation to rupture, and results in an enhancement of the force applied to a bond due to leverage supplied by the bead itself. Experiments yielded median single-bond rupture forces from 25 to 44 pN for IgG from four mammalian species, in general agreement with predictions based on free energies of association obtained from solution equilibrium constants.     

Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers were used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin three-dimensional architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, corresponding to different crossing statuses of the entry/exit DNAs (positive, null or negative, respectively). Torsional strain displaces that equilibrium, leading to an extensive reorganization of the fiber's architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin and may provide the basis to better understand the dynamic binding of chromatin-associated proteins.Note: In the supplementary information initially published online to accompany this article, Supplementary Figure 2 was mistakenly replaced by Supplementary Equation 2. The error has been corrected online.     

Chromatin under mechanical stress: from single 30 nm fibers to single nucleosomes

About a decade ago, the elastic properties of a single chromatin fiber and, subsequently, those of a single nucleosome started to be explored using optical and magnetic tweezers. These techniques have allowed direct measurements of several essential physical parameters of individual nucleosomes and nucleosomal arrays, including the forces responsible for the maintenance of the structure of both the chromatin fiber and the individual nucleosomes, as well as the mechanism of their unwinding under mechanical stress. Experiments on the assembly of individual chromatin fibers have illustrated the complexity of the process and the key role of certain specific components. Nevertheless a substantial disparity exists in the data reported from various experiments. Chromatin, unlike naked DNA, is a system which is extremely sensitive to environmental conditions, and studies carried out under even slightly different conditions are difficult to compare directly. In this review we summarize the available data and their impact on our knowledge of both nucleosomal structure and the dynamics of nucleosome and chromatin fiber assembly and organization.     

Intermediate structure between chromatin fibers and chromosome revealed by mechanical stretching and SPM measurement

The morphology of chromosomes (certain rod-shaped structures) is highly reproducible despite the high condensation of chromatin fibers (∼1 mm) into chromosomes (∼1 μm). However, the mechanism underlying the condensation of chromatin fibers into chromosomes is unclear. We assume that investigation of the internal structure of chromosomes will aid in elucidating the condensation process. In order to observe the detailed structure of a chromosome, we stretched a human chromosome by using a micromanipulator and observed its morphology along the stretched region by scanning probe microscopy (SPM). We found that the chromosome consisted of some fibers that were thicker than chromatin fibers. The found fiber was composed of approximately 90-nm-wide beads that were linked linearly. To explore the components of the fiber, we performed immunofluorescence staining of the stretched chromosome. Fluorescence signals of topoisomerase (Topo) IIα, which is known to interact with and support chromatin fibers, and DNA were detected both on the found fiber and beads. Furthermore, after micrococcal nuclease and trypsin treatments, the fibers were found to be mechanically supported by proteins. These results suggest that chromosome comprises an intermediate structure between chromatin fibers and chromosomes.     

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.     

Nonequilibrium fluctuations of mechanically stretched single red blood cells detected by optical tweezers

We study the thermal and out-of-equilibrium mechanical dynamics of single, living human red blood cells (RBCs) by combining two-probe passive and active microrheology techniques. Both experiments were performed quasisimultaneously on the same cell using two identical polystyrene probes, biochemically attached to the cell membrane. We obtained compelling evidence of nonequilibrium fluctuations in the RBCs under physiological condition and without the influence of any external chemicals. The spectral distributions of metabolically driven forces and viscoelastic response were evaluated in the relaxed and stretched states, intended to simulate the varying natural environment of the cells during blood circulation. We found that the internally generated forces are more pronounced in the stretched state, suggesting a stress-dependent RBC activity.     

Dynamic excitations in membranes induced by optical tweezers.

We present the phenomenology of transformations in lipid bilayers that are excited by laser tweezers. A variety of dynamic instabilities and shape transformations are observed, including the pearling instability, expulsion of vesicles, and more exotic ones, such as the formation of passages. Our physical picture of the laser-membrane interaction is based on the generation of tension in the bilayer and loss of surface area. Although tension is the origin of the pearling instability, it does not suffice to explain expulsion of vesicles, where we observe opening of giant pores and creeping motion of bilayers. We present a quantitative theoretical framework to understand most of the observed phenomenology. The main hypothesis is that lipid is pulled into the optical trap by the familiar dielectric effect, is disrupted, and finally is repackaged into an optically unresolvable suspension of colloidal particles. This suspension, in turn, can produce osmotic pressure and depletion forces, driving the observed transformations.     

Fluorimetric multiparameter cell assay at the single cell level fabricated by optical tweezers

A fluorimetric multi-parameter cell sensor at the single cell level is presented which makes it possible to observe the physiological behavior of different cell lines, different physiological parameters, and statistical data at the same time. Different cell types were immobilized at predefined positions with high accuracy using optical tweezers and adhesion promoting surface layers. The process is applicable to both adherent and non-adherent cells. Coating of the immobilization area with mussel adhesive protein was shown to be essential for the process. Intracellular proton and calcium concentrations in different cell classes were simultaneously imaged and the specific activation of T lymphocytes was demonstrated. This method should be especially useful for drug screening due to the small sample volume and high information density.     

DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.     

Compliance of bacterial polyhooks measured with optical tweezers.

In earlier work, a single-beam gradient force optical trap ("optical tweezers") was used to measure the torsional compliance of flagella in wild-type cells of Escherichia coli that had been tethered to glass by a single flagellum. This compliance was nonlinear, exhibiting a torsionally soft phase up to 180 degrees, followed by a torsionally rigid phase for larger angles. Values for the torsional spring constant in the soft phase were substantially less than estimates based on the rigidity determined for isolated flagellar filaments. It was suggested that the soft phase might correspond to wind-up of the flagellar hook, and the rigid phase to wind-up of the stiffer filament. Here, we have measured the torsional compliance of flagella on cells of an E. coli strain that produces abnormally long hooks but no filaments. The small-angle compliance of these cells, as determined from the elastic rebound of the cell body after wind-up and release, was found to be the same as for wild-type cells. This confirms that the small-angle compliance of wild-type cells is dominated by the response of the hook. Hook flexibility is likely to play a useful role in stabilizing the flagellar bundle.     

Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.     

Experimentally manipulating fungi with optical tweezers

A short review of the use of optical tweezers in fungal cell biological research is provided. First, we describe how optical tweezers work. Second, we review how they have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Third, we indicate how optically trapped microbeads can be used for the localized delivery of chemicals or mechanical stimulation to cells, as well as permitting measurements of the growth forces generated by germ tubes. Finally, the effects of optical trapping on fungal cell viability and growth are assessed. Parts of this review were presented at the Mycological Society of Japan (MSJ) / British Mycological Society (BMS) Joint Symposium, “The new generation mycologists in Japan and the UK” held in Chiba, Japan on June 3, 2006.