Mechanobiology focuses on how physical forces and the mechanical properties of proteins, protein assemblies, cells and tissues contribute to signalling, development, cell division, differentiation and sorting, physiology and disease 1-4. On virtually any scale, ranging from organisms 2,4 to components such as organs 5,6 , tissues 3,7 , cells 8-10 , viruses 11,12 , complex extracellular or intracellular architecture (including vesicles, the extracellular matrix or actin network 13,14) or single proteins 15-17 , biological systems respond to mechanical forces and generate mechanical cues. In mechanobiology, living systems are described by cycles of mechanosensation, mechanotransduction and mechanoresponse 2,18. In addition to its state, the functional response of a living system depends on the nature of the mechanical signal, whether it is applied at the nanometre or micrometre scale, for a short or long time, with low or high magnitude, and on whether it is scalar or vectorial. Nanotechnological and microtechnological approaches have enabled tremendous progress in quantifying the mechanical properties of biological systems. The links between mechanical response, morphology and function, however, are conspicuously ill understood. The most widely used approaches to structurally map the mechanical properties and responses of biological systems, ranging from millimetre to sub-nanometre resolution and from micronewton to piconewton sensitivity, are based on atomic force microscopy (AFM) 19,20. In this Review, we survey the exciting developments in AFM-based approaches towards the morphological mapping of a wide variety of mechanical properties and the characterization of the functional response of biological systems under physiologically relevant conditions. We further discuss key challenges and caveats that have to be taken into account to overcome the limitations of AFM-based approaches to more fully describe the mechanical properties of living systems and highlight how complementary techniques can contribute to directly linking the functional responses of complex biological systems to mechanical cues. Characterizing biosystems by AFM The introduction of AFM in 1986 opened the door to imaging and manipulating matter at the atomic, molecular and cellular scales and was central to the nascent nanotechnological revolution 21,22. Of particular importance for the characterization of biological systems, atomic force microscopes can operate in aqueous environments and at physiological temperatures. In an atomic force microscope, a cantilever that is several micrometres long and has a molecularly sharp probe at the end is used to trace the sample topography, detecting
Protein shells that surround the viral genome. Triangulation numbers Classification system, developed by Caspar and Klug, for icosahedral viruses. T-numbers are integers and contain information on the number of protein subunits that make up a capsid. Virions Viral particles, composed of both capsid proteins and the viral genome, that can successfully infect cells.
Bending of DNA is a feature essential to the function of many DNA-binding proteins. Bending angles can be estimated with a variety of techniques, but most directly from images obtained using scanning force microscopy (SFM). Direct measurement of the bending angle using a tangent method often produces angles that deviate significantly from values obtained using other techniques. Here, we describe the application of SFM in combination with simulations of DNA as a means to estimate protein-induced bending angles in a reliable and unbiased fashion. In this manner, we were able to obtain accurate estimates for the bending angles induced by nuclear factor I, octamer-binding transcription factor 1, the human XPC-Rad23B complex.
The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes a homologous duplex DNA segment and invades it 1,2. After strand exchange, the nucleoprotein filament should disassemble in order for the recombination process to complete 3. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here, we have combined optical tweezers with single-molecule fluorescence microscopy and microfluidics 4,5 to reveal that disassembly results from the interplay between ATP hydrolysis and release of the tension stored in the nucleoprotein filament. Applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. Upon relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results imply that tensiondependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this key homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins. Homologous recombination is a vital mechanism that maintains genome integrity by repairing double-strand breaks in DNA, and generates genetic diversity by exchanging DNA between chromosomes during meiosis. The central process in homologous recombination is the strand exchange between homologous DNA segments. Recombinase proteins such as RecA and RAD51 catalyze this process by forming an ATP-dependent helical filament Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The magnetic tweezer is a single molecule manipulation instrument ideally suited to measuring biophysical systems at a constant applied force. The development of a magnetic tweezers with a high-speed camera and GPU-accelerated particle tracking has allowed for the measurement of molecular events at the millisecond time scale. However, as the spatial resolution of the instrument is improved, previously neglected sources of noise start to become limiting, such as: mechanical stability of the sample stage, and coherent light artifacts such as speckle. Here, we isolate the various sources of noise in an attempt to determine the fundamental limit to magnetic tweezer resolution. We use a state-of-the-art high spatial and temporal resolution magnetic tweezer to measure the dynamics of model systems such as DNA hairpins.
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