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

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Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14kbT. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Hepatitis B virus (HBV) is a major human pathogen. In addition to its importance in human health, there is growing interest in adapting HBV and other viruses for drug delivery and other nanotechnological applications. In both contexts, precise biophysical characterization of these large macromolecular particles is fundamental. HBV capsids are unusual in that they exhibit two distinct icosahedral geometries, nominally composed of 90 and 120 dimers with masses of Ϸ3 and Ϸ4 MDa, respectively. Here, a mass spectrometric approach was used to determine the masses of both capsids to within 0.1%. It follows that both lattices are complete, consisting of exactly 180 and 240 subunits. Nanoindentation experiments by atomic-force microscopy indicate that both capsids have similar stabilities. The data yielded a Young's modulus of Ϸ0.4 GPa. This experimental approach, anchored on very precise and accurate mass measurements, appears to hold considerable potential for elucidating the assembly of viruses and other macromolecular particles.atomic force microscopy ͉ collision-induced dissociation ͉ macromolecular mass spectrometry ͉ virus assembly ͉ viral structural biology H epatitis B virus (HBV) is a major cause of liver disease in humans (1), with Ͼ350 million people suffering from chronic infection. For the development of new antiviral drugs, further insight into the replication cycle and assembly pathway of the virus is needed (2). Moreover, there is a growing interest in HBV and other viral particles as vehicles for drug delivery and as platforms for nanoparticle technology (3). In this context, precise biophysical characterization of these particles represents essential basic information.HBV has an enveloped virion. Single-stranded viral RNA is packaged into the assembling capsid and, within this compartment, is reverse-transcribed into DNA (4, 5). The DNAcontaining nucleocapsid then proceeds to envelopment. Both in vivo and in vitro, the capsid protein (cp) forms icosahedral capsids of two sizes, corresponding to triangulation numbers of T ϭ 3 and T ϭ 4 (6), nominally consisting of 180 and 240 subunits, respectively (7-10). Cp has a 140-residue N-terminal core domain connected to a 34-residue ''protamine domain'' by a 10-residue linker (11). The protamine domain binds RNA, whereas the core domain is necessary and sufficient for capsid assembly. The ratio of T ϭ 3 to T ϭ 4 capsids produced depends on the length of the linker and the conditions of assembly: The smaller T ϭ 3 capsid becomes progressively more abundant as the linker is shortened (12). The building-block for capsid formation is a dimer stabilized via an intermolecular four-helix bundle (13-15) and a disulfide bond within the bundle (Cys61). However, dimerization and assembly also occur in the absence of the disulfide, e.g., when Cys61 is replaced with Ala (10, 12). The capsid has protruding spikes at the dimer interfaces that display most of the antigenic epitopes and holes at the symmetry axes that allow infusion of nucleotides for reverse transcription (7, ...