Nonlinear mechanics of human mitotic chromosomes.

In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped chromosomes1. This metamorphosis is driven mainly by the combined action of condensins and topoisomerase IIα (TOP2A)2,3, and has been observed using microscopy for over a century. Nevertheless, very little is known about the structural organization of a mitotic chromosome. Here we introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behaviour, distinct from that predicted by classical polymer models4. To explain this anomalous stiffening, we introduce a hierarchical worm-like chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A5 specifically in mitosis, we provide evidence that TOP2A has a role in the preservation of chromosome compaction. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.

Regulation of T7 gp2.5 binding dynamics by its C-terminal tail, template conformation and sequence.

Bacteriophage T7 single-stranded DNA-binding protein (gp2.5) binds to and protects transiently exposed regions of single-stranded DNA (ssDNA) while dynamically interacting with other proteins of the replication complex. We directly visualize fluorescently labelled T7 gp2.5 binding to ssDNA at the single-molecule level. Upon binding, T7 gp2.5 reduces the contour length of ssDNA by stacking nucleotides in a force-dependent manner, suggesting T7 gp2.5 suppresses the formation of secondary structure. Next, we investigate the binding dynamics of T7 gp2.5 and a deletion mutant lacking 21 C-terminal residues (gp2.5-Δ21C) under various template tensions. Our results show that the base sequence of the DNA molecule, ssDNA conformation induced by template tension, and the acidic terminal domain from T7 gp2.5 significantly impact on the DNA binding parameters of T7 gp2.5. Moreover, we uncover a unique template-catalyzed recycling behaviour of T7 gp2.5, resulting in an apparent cooperative binding to ssDNA, facilitating efficient spatial redistribution of T7 gp2.5 during the synthesis of successive Okazaki fragments. Overall, our findings reveal an efficient binding mechanism that prevents the formation of secondary structures by enabling T7 gp2.5 to rapidly rebind to nearby exposed ssDNA regions, during lagging strand DNA synthesis.

Viscoelastic phenotyping of red blood cells.

Red blood cells (RBCs) are the simplest cell types with complex dynamical and viscoelastic phenomenology. While the mechanical rigidity and the flickering noise of RBCs have been extensively investigated, an accurate determination of the constitutive equations of the relaxational kinetics is lacking. Here we measure the force relaxation of RBCs under different types of tensional and compressive extension-jump protocols by attaching an optically trapped bead to the RBC membrane. Relaxational kinetics follows linear response from 60 pN (tensional) to -20 pN (compressive) applied forces, exhibiting a triple exponential function with three well-separated timescales over four decades (0.01-100 s). While the fast timescale (τF∼0.02(1)s) corresponds to the relaxation of the membrane, the intermediate and slow timescales (τI=4(1)s; τS=70(8)s) likely arise from the cortex dynamics and the cytosol viscosity. Relaxation is highly heterogeneous across the RBC population, yet the three relaxation times are correlated, showing dynamical scaling. Finally, we find that glucose depletion and laser illumination of RBCs lead to faster triple exponential kinetics and RBC rigidification. Viscoelastic phenotyping is a promising dynamical biomarker applicable to other cell types and active systems.

Control of septum thickness by the curvature of SepF polymers.

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF. We have isolated SepF homologs from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 44 nm. Interestingly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF regulates ring diameter. Importantly, when SepF chimeras with different diameters were expressed in the bacterial host Bacillus subtilis, the thickness of its septa changed accordingly. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mold to shape the cell wall.

Unravelling the mechanisms of Type 1A topoisomerases using single-molecule approaches.

Topoisomerases are essential enzymes that regulate DNA topology. Type 1A family topoisomerases are found in nearly all living organisms and are unique in that they require single-stranded (ss)DNA for activity. These enzymes are vital for maintaining supercoiling homeostasis and resolving DNA entanglements generated during DNA replication and repair. While the catalytic cycle of Type 1A topoisomerases has been long-known to involve an enzyme-bridged ssDNA gate that allows strand passage, a deeper mechanistic understanding of these enzymes has only recently begun to emerge. This knowledge has been greatly enhanced through the combination of biochemical studies and increasingly sophisticated single-molecule assays based on magnetic tweezers, optical tweezers, atomic force microscopy and Förster resonance energy transfer. In this review, we discuss how single-molecule assays have advanced our understanding of the gate opening dynamics and strand-passage mechanisms of Type 1A topoisomerases, as well as the interplay of Type 1A topoisomerases with partner proteins, such as RecQ-family helicases. We also highlight how these assays have shed new light on the likely functional roles of Type 1A topoisomerases in vivo and discuss recent developments in single-molecule technologies that could be applied to further enhance our understanding of these essential enzymes.

Unravelling How Single-Stranded DNA Binding Protein Coordinates DNA Metabolism Using Single-Molecule Approaches.

Single-stranded DNA-binding proteins (SSBs) play vital roles in DNA metabolism. Proteins of the SSB family exclusively and transiently bind to ssDNA, preventing the DNA double helix from re-annealing and maintaining genome integrity. In the meantime, they interact and coordinate with various proteins vital for DNA replication, recombination, and repair. Although SSB is essential for DNA metabolism, proteins of the SSB family have been long described as accessory players, primarily due to their unclear dynamics and mechanistic interaction with DNA and its partners. Recently-developed single-molecule tools, together with biochemical ensemble techniques and structural methods, have enhanced our understanding of the different coordination roles that SSB plays during DNA metabolism. In this review, we discuss how single-molecule assays, such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations, have advanced our understanding of the binding dynamics of SSBs to ssDNA and their interaction with other proteins partners. We highlight the central coordination role that the SSB protein plays by directly modulating other proteins' activities, rather than as an accessory player. Many possible modes of SSB interaction with protein partners are discussed, which together provide a bigger picture of the interaction network shaped by SSB.

Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA.

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4-XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations.

Imaging unlabeled proteins on DNA with super-resolution.

Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or 'iBALM' can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

Supercoiling DNA optically.

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

PICH: a DNA translocase specially adapted for processing anaphase bridge DNA.

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms.

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

Acetylation and phosphorylation of human TFAM regulate TFAM-DNA interactions via contrasting mechanisms.

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression and transmission of mitochondrial DNA (mtDNA). However, mechanisms for the post-translational regulation of TFAM are poorly understood. Here, we show that TFAM is lysine acetylated within its high-mobility-group box 1, a domain that can also be serine phosphorylated. Using bulk and single-molecule methods, we demonstrate that site-specific phosphoserine and acetyl-lysine mimics of human TFAM regulate its interaction with non-specific DNA through distinct kinetic pathways. We show that higher protein concentrations of both TFAM mimics are required to compact DNA to a similar extent as the wild-type. Compaction is thought to be crucial for regulating mtDNA segregation and expression. Moreover, we reveal that the reduced DNA binding affinity of the acetyl-lysine mimic arises from a lower on-rate, whereas the phosphoserine mimic displays both a decreased on-rate and an increased off-rate. Strikingly, the increased off-rate of the phosphoserine mimic is coupled to a significantly faster diffusion of TFAM on DNA. These findings indicate that acetylation and phosphorylation of TFAM can fine-tune TFAM-DNA binding affinity, to permit the discrete regulation of mtDNA dynamics. Furthermore, our results suggest that phosphorylation could additionally regulate transcription by altering the ability of TFAM to locate promoter sites.

Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level.

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.

Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU.

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU-DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence.

The ability to measure mechanics and forces in biological nanostructures, such as DNA, proteins and cells, is of great importance as a means to analyze biomolecular systems. However, current force detection methods often require specialized instrumentation. Here, we present a novel and versatile method to quantify tension in molecular systems locally and in real time, using intercalated DNA fluorescence. This approach can report forces over a range of at least ∼0.5-65 pN with a resolution of 1-3 pN, using commercially available intercalating dyes and a general-purpose fluorescence microscope. We demonstrate that the method can be easily implemented to report double-stranded (ds)DNA tension in any single-molecule assay that is compatible with fluorescence microscopy. This is particularly useful for multiplexed techniques, where measuring applied force in parallel is technically challenging. Moreover, tension measurements based on local dye binding offer the unique opportunity to determine how an applied force is distributed locally within biomolecular structures. Exploiting this, we apply our method to quantify the position-dependent force profile along the length of flow-stretched DNA and reveal that stretched and entwined DNA molecules-mimicking catenated DNA structures in vivo-display transient DNA-DNA interactions. The method reported here has obvious and broad applications for the study of DNA and DNA-protein interactions. Additionally, we propose that it could be employed to measure forces in any system to which dsDNA can be tethered, for applications including protein unfolding, chromosome mechanics, cell motility, and DNA nanomachines.

Effect of dsDNA on the Assembly Pathway and Mechanical Strength of SV40 VP1 Virus-like Particles.

Simian virus 40 (SV40) is a possible vehicle for targeted drug delivery systems because of its low immunogenicity, high infectivity, and high transfection efficiency. To use SV40 for biotechnology applications, more information is needed on its assembly process to efficiently incorporate foreign materials and to tune the mechanical properties of the structure. We use atomic force microscopy to determine the effect of double-stranded DNA packaging, buffer conditions, and incubation time on the morphology and strength of virus-like particles (VLPs) composed of SV40 VP1 pentamers. DNA-induced assembly results in a homogeneous population of native-like, ∼45 nm VLPs. In contrast, under high-ionic-strength conditions, the VP1 pentamers do not seem to interact consistently, resulting in a heterogeneous population of empty VLPs. The stiffness of both in-vitro-assembled empty and DNA-filled VLPs is comparable. Yet, the DNA increases the VLPs' resistance to large deformation forces by acting as a scaffold, holding the VP1 pentamers together. Both disulfide bridges and Ca2+, important in-vitro-assembly factors, affect the mechanical stability of the VLPs: the reducing agent DTT makes the VLPs less resistant to mechanical stress and prone to damage, whereas Ca2+-chelating EDTA induces a marked softening of the VLP. These results show that negatively charged polymers such as DNA can be used to generate homogeneous particles, thereby optimizing VLPs as vessels for drug delivery. Moreover, the storage buffer should be chosen such that VP1 interpentamer interactions are preserved.

Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways.

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

Acoustic force spectroscopy.

Force spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Here we extend the force spectroscopy toolbox with an acoustic manipulation device that can exert forces from subpiconewtons to hundreds of piconewtons on thousands of biomolecules in parallel, with submillisecond response time and inherent stability. This method can be readily integrated in lab-on-a-chip devices, allowing for cost-effective and massively parallel applications.

Vertex-Specific Proteins pUL17 and pUL25 Mechanically Reinforce Herpes Simplex Virus Capsids.

Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of icosahedral herpes simplex virus capsids. pUL17 and pUL25, which form the capsid vertex-specific component (CVSC), particularly contributed to capsid resilience along the 5-fold and 2-fold but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry of the protein composition of the capsids, revealed that both pUL17 and pUL25 are required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids.IMPORTANCE In this study, the structural, material properties of herpes simplex virus 1 were investigated. The capsid of herpes simplex virus is built up of a variety of proteins, and we scrutinized the influence of two of these proteins on the stability of the capsid. For this, we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach, we revealed that both studied proteins play an essential role in viral stability. These new insights support us in forming a complete view on viral structure and furthermore could possibly help not only to develop specific antivirals but also to build protein shells with improved stability for drug delivery purposes.