During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a singlemolecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.ouble-stranded DNA (dsDNA) breaks are severe forms of genetic lesions that may result in chromosome instability (1-3). Organisms have devised several pathways to mend dsDNA breaks. Among these, recombinational repair mediated by bacterial RecA-like ATP-dependent recombinases is the most accurate, because it is capable of restoring chromosome integrity without loss of genetic information (2, 4). During recombinational repair in humans, broken dsDNA ends are first resected to create single-stranded DNA (ssDNA) overhangs that are coated quickly by replication protein A (RPA). The ATP-dependent recombinase protein RAD51, the focus of this study, must next compete with RPA to assemble nucleoprotein filaments around these ssDNA overhangs. These filaments form the structures that can promote homology recognition in an intact homologous duplex and catalyze DNA strand exchange, resulting in joint molecule intermediates. After RAD51 disassembly from the heteroduplex DNA, the invading strand can prime DNA synthesis to recover lost genetic information. RAD51, however, does not act alone during recombinational repair. Mediators and accessory factors stringently control the dynamics of RAD51 filaments by acting at the level of formation, stabilization, or even disassembly of these filaments (2,3,5). One important level of control occurs at the assembly of nascent RAD51 filaments on RPA-coated ssDNA. On its own, RAD51 cannot load on the RPA-coated substrate but requires the action of a mediator to guide an...

Structure and function of viruses are intimately related, and one of the goals in virology is to elucidate the mechanisms behind this relation. A variety of research endeavours is focused on studying these mechanisms and a relatively new technique in this field is Atomic Force Microscopy (AFM). Using AFM virions and virus-like particles can be imaged and manipulated at the single particle level. Here we review recent AFM nano-indentations studies unveiling for instance the mechanics of capsid-genome interactions, morphological changes that drive viral maturation, capsid stabilizing factors and viral uncoating. We show that in an increasing amount of literature a clear link between mechanics and infectivity is observed, which not only provides us with new fundamental insights into virology, but also provides ways to improve virus-like particles for applications in nanomedicine and nanotechnology.

Small multilamellar vesicles may have benefits over unilamellar vesicles for drug delivery, such as an increased volume for hydrophobic drugs. In addition, their altered mechanical properties might be beneficial for cellular uptake. Here, we show how atomic force microscopy (AFM) can be used to detect and characterize multilamellar vesicles. We quantify the size of each break event occurring during AFM nanoindentations, which shows good agreement with the thickness of supported lipid bilayers. Analyzing the size and number of these events for individual vesicles allows us to distinguish between vesicles consisting of 1 up to 5 bilayers. We validate these results by comparison with correlative cryo-electron microscopy (cryo-EM) data at the vesicle population level. Finally, we quantify the vesicle geometry and mechanical properties, and show that with additional bilayers adherent vesicles are more spherical and stiffer. Surprisingly, at ∼20% stiffening for each additional bilayer, the vesicle stiffness scales only weakly with lamellarity. Our results show the potential of AFM for studying liposomal nanoparticles and suggest that small multilamellar vesicles may have beneficial mechanical properties for cellular uptake.

While the structure of a multitude of viral particles has been resolved to atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues are particle nucleation, particle growth, and the mode of genome compaction. These issues are difficult to address in bulk approaches and are effectively only accessible by the real-time tracking of assembly dynamics of individual particles. This we do here by studying the assembly into rod-shaped viruslike particles (VLPs) of artificial capsid polypeptides. Using fluorescence optical tweezers, we establish that small oligomers perform one-dimensional diffusion along the DNA. Larger oligomers are immobile and nucleate VLP growth. A multiplexed acoustic force spectroscopy approach reveals that DNA is compacted in regular steps, suggesting packaging via helical wrapping into a nucleocapsid. By reporting how real-time assembly tracking elucidates viral nucleation and growth principles, our work opens the door to a fundamental understanding of the complex assembly pathways of both VLPs and naturally evolved viruses.