A master relation defines the nonlinear viscoelasticity of single fibroblasts

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 200

A versatile maskless microscope projection photolithography system and its application in light-directed fabrication of DNA microarrays

We present a maskless microscope projection lithography system (MPLS), in which photomasks have been replaced by a Digital Micromirror Device type spatial light modulator (DMD, Texas Instruments).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 articl

Bead-based assay for spatiotemporal gene expression control in cell-free transcription-translation systems

Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space

DNA oligomer binding in competition exhibits cooperativity

Binding of two complementary DNA single strands to a double-helix, DNA hybridization, is a sequence specific molecular recognition process that plays important roles in biology and biotechnological applications. In the past much work has been devoted to understand double helix formation, however, DNA binding in complex situations often remains difficult to deal with. Here we use fluorescence anisotropy to assess the binding affinities of DNA oligonucleotide strands that compete for hybridization to the same probe molecule in thermal equilibrium. We find that the ratio of the binding constants in competition can change substantially compared to pairwise assessments. This is a signature of non-trivial interaction among the competitors: the binding microstates of each strand are affected by the presence of the other, but to a different degree. To our knowledge this type of phenomenon is not included in current equilibrium models of oligonucleotide binding. We suggest interactions beyond double helix conformations to cause the observed cooperative behavior. The cooperativity could produce more complex binding phenomena than previously thought

Hydra Molecular Network Reaches Criticality at the Symmetry-Breaking Axis-Defining Moment

We study biological, multicellular symmetry breaking on a hollow cell sphere as it occurs during hydra regeneration from a random cell aggregate. We show that even a weak temperature gradient directs the axis of the regenerating animal but only if it is applied during the symmetry-breaking moment. We observe that the spatial distribution of the early expressed, head-specific gene k s 1 has become scale-free and fractal at that point. We suggest the self-organized critical state to reflect long range signaling, which is required for axis definition and arises from cell next-neighbor communication

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of L-lysine by L-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational L-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinke

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins