Biophysical Reviews and Letters

Protein–DNA loops are essential for efficient transcriptional repression and activation. The geometry and stability of the archetypal Lac repressor tetramer (LacI)–DNA loop were investigated using designed hyperstable loops containing lac operators bracketing a sequence-directed bend. Electrophoretic mobility shift assays, DNA cyclization, and bulk and single-molecule fluorescence resonance energy transfer (FRET) demonstrate that the DNA sequence controls whether the LacI–DNA loop forms a compact loop with positive writhe or an open loop with little writhe. Monte Carlo methods for simulation of DNA ring closure were extended to DNA loops, including treatment of variable protein hinge angles. The observed distribution of topoisomer products upon cyclization provides a strong constraint on possible models. The experiments and modeling imply that LacI–DNA can adopt a wide range of geometries but has a strong intrinsic preference for an open form. The flexibility of LacI helps explain in vivo observations that DNA looping is less sensitive to DNA length and shape than that expected from the physical properties of DNA. While DNA cyclization suggests two pools of precursor loops for the 9C14 construct, single-molecule FRET demonstrates a single population. This discrepancy suggests that the LacI–DNA structure is strongly influenced by flanking DNA.

We theoretically study the role of enzyme-induced bending in DNA-cleavage processes. In our model the enzyme bends DNA at its adsorption site leading to stress propagation along the filaments. Since this process is slow the time-dependent DNA-shape can be calculated in a quasistatic approximation. This allows to determine the range of parameters for which strong DNA bending occurs. For enzymes which cut by bending only the cleavage rate is found to depend crucially on the viscosity of the surrounding fluid and the length of the cut DNA segment.

Molecular motors power directed transport of cargoes within cells. Even if a single motor is sufficient to transport a cargo, motors often cooperate in small teams. We discuss the cooperative cargo transport by several motors theoretically and explore some of its properties. In particular we emphasize how motor teams can drag cargoes through a viscous environment.

We study the cooperative behavior of cytoskeletal filaments in motility assays, in which immobilized motor proteins bind the filaments to a surface and actively pull them along this surface. Because of the repulsive interaction of filaments, the motor-driven dynamics of filaments leads to a nonequilibrium phase transition which generalizes the isotropicnematic phase transition of the corresponding equilibrium system, the hard-rod fluid. Langevin dynamics simulations and analytical theory show that the motor activity enhances the tendency for nematic ordering. At high detachment forces of motors, we observe the formation of filament clusters because of blocking effects; at low detachment forces, cluster formation can be controlled by the density of inactive motors.

We discuss how translocation properties of the 20S proteasome influence its length distribution, one of its most important feature for the normal functioning of the immune system. For this we consider a simple one-channel proteasome model and assume that the protein transport depends significantly on the length of a protein located inside the proteasome chamber. Using the master equation approach we show analytically that the length distribution with one dominating peak, observed in the experiments, can be achieved if the transport rate function is in a certain relation with cleavage probabilities and the geometry of a proteasome. Our analytical results are confirmed by numerical simulations of the protein degradation by the proteasome performed using the modified Gillespie algorithm.

The interaction of electric fields with lipid membranes and cells has been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest because of their widespread use in cell biology and biotechnology. Giant vesicles, being of cell size and convenient for microscopy observations, are the simplest model of the cell membrane. However, optical microscopy observation of effects caused by electric DC pulses on giant vesicles is difficult because of the short duration of the pulse. Recently this difficulty has been overcome in our lab. Using a digital camera with high temporal resolution, we were able to access vesicle fusion dynamics on a sub-millisecond time scale. In this report, we present some observations on electrodeformation and –poration of single vesicles followed by an extensive study on the electrofusion of vesicle couples. Finally, we suggest an attractive approach for creating multidomain vesicles using electrofusion and present some preliminary results on the effect of membrane stiffness on the fusion dynamics.

Most biological tissues are soft viscoelastic materials with elastic moduli ranging from approximately 100 to 100,000 Pa. Recent studies have examined the effect of substrate rigidity on cell structure and function, and many, but not all cell types exhibit a strong response to substrate stiffness. Some blood cells such as platelets and neutrophils have indistinguishable structures on hard and soft materials as long as they are sufficiently adhesive, whereas many cell types, including fibroblasts and endothelial cells spread much more strongly on rigid compared to soft substrates. A few cell types such as neurons appear to extend better on very soft materials. The different response of astrocytes and neurons to the stiffness of their substrate results in preferential growth of neurons on soft gels and astrocytes on hard gels, and suggests that preventing rigidification of damaged central nervous system tissue after injury may have utility in wound healing. How cells sense substrate stiffness is unknown. One candidate protein, filamin A, which responds to externally derived stresses, was tested in melanoma cells. Cells devoid of filamin A retain the ability to sense substrate stiffness, suggesting that other proteins are required for stiffness sensing.

Optical tweezers are a versatile tool in biophysics and have matured from a tool of manipulation to a tool of precise measurements. We argue here that the data analysis with advantage can be developed to a level of sophistication that matches that of the instrument. We review methods of analysis of optical tweezers data, primarily based on the power spectra of time series of positions for trapped spherical objects. The majority of precise studies in the literature are performed on in vitro systems, whereas in the present work, an example of an in vivo system is presented for which precise power spectral analysis is both useful and necessary. The biological system is the cytoplasm of fission yeast, Schizosaccharomyces pombe in which we observe subdiffusion of lipid granules. In a search for the cause of subdiffusion, we chemically disrupt the actin network in the cytoplasm and further consider in vitro networks of filamenteous actin undergoing similar chemical disruption.

In the course of our investigations of routes for general and convenient synthesis of amphiphiles derived from sugars, we reported new synthetic ways to prepare catanionic surfactants, polymers and dendrimers using unprotected lactose or lactobionic acid. Moreover we developed amphiphilic dendrimers bearing sugar polar heads. These compounds are of interest for their biological applications (mimics of natural ligands of proteins (e.g. gp 120 of HIV), encapsulation, vectorisation).

Poly(acrylic acid) (PAAc) is a commonly used polymer grafted on poly(ethylene terephthalate) films for the immobilization of bioactive molecules that directly interact with living cells or tissues for the maintenance of their viability and functionality. The diffusion property of the grafted PAAc on the surface is a critical parameter related to the density, length of polymer chains, and ionic strength of the solution. Fluorescence correlation spectroscopy (FCS) is able to measure the diffusion coefficient of fluorescent particles in solution with single molecule sensitivity and specificity. It was used as an effective tool to detect diffusion dynamics of Atto 565 molecules, a good indicator for viscosity of PAAc, in both aqueous polymer solutions and polymer grafted film surfaces immersed in solution. In this work we determine the polymer chain length under different polymerization conditions in solution and deduce the solution viscosity by FCS measurements of Atto 565 as tracer molecule. By using the same tracer on the grafted polymer films we can infer the viscosity of these grafted layers under a variety of conditions, including the PAAc chain length, the UV exposure time during polymerization, the ionic strength, and the pH value of the immersed solution.

Cell-based approaches in medicine, biotechnology and in pharmaceutical research offer unique prospects to cope with future challenges in the field of public health. Stem cell research, autologous cell therapies and tissue engineering are only a few possible key applications. Progress in these fields will depend on the successful implementation of versatile and flexible tools for the gentle manipulation and characterization of cells. In recent years, we and others have introduced microfluidic lab-on-chip systems that include dielectrophoretic elements for the contact-less handling and the analysis of cells. Here, we present results that were obtained by combining our labon-on-chip devices with a low-cost centrifugation stage for the efficient and gentle separation of microparticles and live human cells. Our approach is supposed to overcome limitations that arise from the use of bulky and expensive external pumping stages.