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Many cellular processes are tightly connected to the dynamics of microtubules (MTs). While in neuronal axons MTs mainly regulate intracellular trafficking, they participate in cytoskeleton reorganization in many other eukaryotic cells, enabling the cell to efficiently adapt to changes in the environment. We show that the functional differences of MTs in different cell types and regions is reflected in the dynamic properties of MT tips. Using plus-end tracking proteins EB1 to monitor growing MT plus-ends, we show that MT dynamics and life cycle in axons of human neurons significantly differ from that of fibroblast cells. The density of plus-ends, as well as the rescue and catastrophe frequencies increase while the growth rate decreases toward the fibroblast cell margin. This results in a rather stable filamentous network structure and maintains the connection between nucleus and membrane. In contrast, plus-ends are uniformly distributed along the axons and exhibit diverse polymerization run times and spatially homogeneous rescue and catastrophe frequencies, leading to MT segments of various lengths. The probability distributions of the excursion length of polymerization and the MT length both follow nearly exponential tails, in agreement with the analytical predictions of a two-state model of MT dynamics., Comment: 10 pages, 4 figures

The specificity of molecular recognition is important to molecular self-organization. A prominent example is the biological cell where, within a highly crowded molecular environment, a myriad of different molecular receptor pairs recognize their binding partner with astonishing accuracy. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide strand can prevail against a disproportionally more concentrated competitor that exhibits reduced affinity due to a mismatch. The magnitude of deviation from the simple picture above may reach several orders of magnitude. In our experiments the effective molecular affinity of a given strand remains elevated only as long as the better matching competitor is not present. We interpret our observations based on an energy-barrier of entropic origin that occurs if two competing oligonucleotide strands occupy the same probe simultaneously. In this situation the relative binding affinities are reduced asymmetrically, which leads to an expression of the free energy landscape that represents a formal analogue of a Landau description of phase transitions. Our mean field description reproduces the observations in quantitative agreement. The advantage of improved molecular recognition comes at no energetic cost other than the design of the molecular ensemble, and the introduction of the competitor. It will be interesting to see if mechanisms along similar lines as exposed here, contribute to the molecular synergy that occurs in biological systems.

Cell mechanical properties are fundamental to the organism but remain poorly understood. We report a comprehensive phenomenological framework for the nonlinear rheology of single fibroblast cells: a superposition of elastic stiffening and viscoplastic kinematic hardening. Our results show, that in spite of cell complexity its mechanical properties can be cast into simple, well-defined rules, which provide mechanical cell strength and robustness via control of crosslink slippage., Comment: 4 pages, 6 figures

Microarray-based genotyping is based on the high discrimination capability of oligonucleotide probes. For detection of Single Nucleotide Polymorphisms (SNPs) single-base discrimination is required. We investigate how various point-mutations, comprising single base mismatches (MMs), insertions and deletions, affect hybridization of DNA-DNA oligonucleotide duplexes. Employing light-directed in situ synthesis we fabricate DNA microarrays with comprehensive sets of cognate point-mutated probes, allowing us to systematically investigate the influence of defect type, position and nearest neighbor effects. Defect position has been identified as the dominating influential factor. This positional effect which is almost identical for the different point-mutation types, is biased from the local sequence environment. The impact of the MM type is largely determined by the type of base pair (either AT or CG) affected by the mismatch. We observe that single base insertions next to like-bases result in considerably larger hybridization signals than insertions next to nonidentical bases. The latter as well as the distinct position dependence could be explained by a kinetic zipper model in which point defects represent a barrier for the rapid closure of the DNA duplex., Comment: 12 pages, 12 figures

We present a maskless microscope projection lithography system (MPLS), in which photomasks have been replaced by a Digital Micromirror Device type spatial light modulator (DMD, Texas Instruments). Employing video projector technology high resolution patterns, designed as bitmap images on the computer, are displayed using a micromirror array consisting of about 786000 tiny individually addressable tilting mirrors. The DMD, which is located in the image plane of an infinity corrected microscope, is projected onto a substrate placed in the focal plane of the microscope objective. With a 5x(0.25 NA) Fluar microscope objective, a fivefold reduction of the image to a total size of 9 mm2 and a minimum feature size of 3.5 microns is achieved. Our system can be used in the visible range as well as in the near UV (with a light intensity of up to 76 mW/cm2 around the 365 nm Hg-line). We developed an inexpensive and simple method to enable exact focusing and controlling of the image quality of the projected patterns. Our MPLS has originally been designed for the light-directed in situ synthesis of DNA microarrays. One requirement is a high UV intensity to keep the fabrication process reasonably short. Another demand is a sufficient contrast ratio over small distances (of about 5 microns). This is necessary to achieve a high density of features (i.e. separated sites on the substrate at which different DNA sequences are synthesized in parallel fashion) while at the same time the number of stray light induced DNA sequence errors is kept reasonably small. We demonstrate the performance of the apparatus in light-directed DNA chip synthesis and discuss its advantages and limitations., Comment: 12 pages, 9 figures, journal article

Cell mechanical functions like locomotion, contraction and division are controlled by the cytoskeleton, a dynamic biopolymer network whose mechanical properties remain poorly understood. We perform single-cell uniaxial stretching experiments on 3T3 fibroblasts. By superimposing small amplitude oscillations on a mechanically prestressed cell, we find a transition from linear viscoelastic behavior to power-law stress stiffening. Data from different cells over several stress decades can be uniquely scaled to obtain a master-relation between the viscoelastic moduli and the average force. Remarkably, this relation holds independently of deformation history, adhesion biochemistry, and intensity of active contraction. In particular, it is irrelevant whether force is actively generated by the cell or externally imposed by stretching. We propose that the master-relation reflects the mechanical behavior of the force bearing actin cytoskeleton, in agreement with stress stiffening known from semiflexible filament networks., Comment: 12 pages, 11 figures. Accepted for publication in Biophysical Journal, scheduled to appear in May 2006

We report a cylindrical-peristaltic shape transformation in axons exposed to a controlled osmotic perturbation. The peristaltic shape relaxes and the axon recovers its original geometry within minutes. We show that the shape instability depends critically on swelling rate and that volume and membrane area regulation are responsible for the shape relaxation. We propose that volume regulation occurs via leakage of ions driven by elastic pressure, and analyse the peristaltic shape dynamics taking into account the internal structure of the axon. The results obtained provide a framework for understanding peristaltic shape dynamics in nerve fibers occurring in vivo., Comment: 4 pages, 4 figures. Accepted for publication in Physical Review Letters

We present a general theoretical analysis of semiflexible filaments subject to viscous drag or point forcing. These are the relevant forces in dynamic experiments designed to measure biopolymer bending moduli. By analogy with the ``Stokes problems" in hydrodynamics (fluid motion induced by that of a wall bounding a viscous fluid), we consider the motion of a polymer one end of which is moved in an impulsive or oscillatory way. Analytical solutions for the time-dependent shapes of such moving polymers are obtained within an analysis applicable to small-amplitude deformations. In the case of oscillatory driving, particular attention is paid to a characteristic length determined by the frequency of oscillation, the polymer persistence length, and the viscous drag coefficient. Experiments on actin filaments manipulated with optical traps confirm the scaling law predicted by the analysis and provide a new technique for measuring the elastic bending modulus. A re-analysis of several published experiments on microtubules is also presented., Comment: RevTex, 24 pages, 15 eps figs, uses cite.sty, Biophysical J