Viscoelasticity of diverse biological samples quantified by Acoustic Force Microrheology (AFMR).

In the context of soft matter and cellular mechanics, microrheology - the use of micron-sized particles to probe the frequency-dependent viscoelastic response of materials - is widely used to shed light onto the mechanics and dynamics of molecular structures. Here we present the implementation of active microrheology in an Acoustic Force Spectroscopy setup (AFMR), which combines multiplexing with the possibility of probing a wide range of forces ( ~ pN to ~nN) and frequencies (0.01-100 Hz). To demonstrate the potential of this approach, we perform active microrheology on biological samples of increasing complexity and stiffness: collagen gels, red blood cells (RBCs), and human fibroblasts, spanning a viscoelastic modulus range of five orders of magnitude. We show that AFMR can successfully quantify viscoelastic properties by probing many beads with high single-particle precision and reproducibility. Finally, we demonstrate that AFMR to map local sample heterogeneities as well as detect cellular responses to drugs.

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

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.

Microfluidics and fluorescence microscopy protocol to study the response of C. elegans to chemosensory stimuli.

Here, we present a protocol to use microfluidics in combination with fluorescence microscopy to expose the C. elegans tail to chemosensory stimuli. We describe steps for the preparation of microfluidic chips and sample preparation through the sedation of C. elegans. We detail flow calibration and imaging of C. elegans through fluorescence microscopy to determine their molecular and/or cellular response to chemosensory stimuli. This protocol can also be applied to amphid neurons by inserting the worm in the chip head-first. For complete details on the use and execution of this protocol, please refer to Bruggeman et al. (2022).1.

PICH acts as a force-dependent nucleosome remodeler.

In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. Here, we present an in vitro single-molecule assay that mimics chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.

Differentiated dynamic response in C. elegans chemosensory cilia.

Cilia are membrane-enveloped organelles that protrude from the surface of most eurokaryotic cells and play crucial roles in sensing the external environment. For maintenance and function, cilia are dependent on intraflagellar transport (IFT). Here, we use a combination of microfluidics and fluorescence microscopy to study the response of phasmid chemosensory neurons, in live Caenorhabditis elegans, to chemical stimuli. We find that chemical stimulation results in unexpected changes in IFT and ciliary structure. Notably, stimulation with hyperosmotic solutions or chemical repellents results in different responses, not only in IFT, ciliary structure, and cargo distribution, but also in neuronal activity. The response to chemical repellents results in habituation of the neuronal activity, suggesting that IFT plays a role in regulating the chemosensory response. Our findings show that cilia are able to sense and respond to different external cues in distinct ways, highlighting the flexible nature of cilia as sensing hubs.

Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System.

Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the versatility of optical trapping. Quadruple (Q)-trap optical tweezers are an excellent example of such an advance, by providing three-dimensional control over two constructs and thereby enabling for example DNA-DNA braiding. However, the implementation of fluorescence detection in such a Q-trapping system poses several challenges: (1) since typical samples span a distance in the order of tens of micrometers, it requires imaging of a large field of view, (2) in order to capture fast molecular dynamics, fast imaging with single-molecule sensitivity is desired, (3) in order to study three-dimensional objects, it could be needed to detect emission light at different axial heights while keeping the objective lens and thus the optically trapped microspheres in a fixed position. In this chapter, we describe design guidelines for a fluorescence imaging module on a Q-trap system that overcomes these challenges and provide a step-by-step description for construction and alignment of such a system. Finally, we present detailed instructions for proof-of-concept experiments that can be used to validate and highlight the capabilities of the instruments.

One-Dimensional STED Microscopy in Optical Tweezers.

Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.

Temperature Quantification and Temperature Control in Optical Tweezers.

Optical tweezers are widely used to investigate biomolecules and biomolecular interactions. In these investigations, the biomolecules of interest are typically coupled to microscopic beads that can be optically trapped. Since high-intensity laser beams are required to trap such microscopic beads, laser-induced heating due to optical absorption is typically unavoidable. This chapter discusses how to identify, quantify, and control thermal effects in optical tweezers. We provide a brief overview of the reported causes and effects of unwanted heating in optical tweezers systems. Specific details are provided on methods to perform a temperature-independent trap calibration procedure. Finally, an effective temperature-control system is presented, and we discuss the operation of this system as well as the methods to measure the temperature at the optically trapped particle.

Generating Negatively Supercoiled DNA Using Dual-Trap Optical Tweezers.

Many genomic processes lead to the formation of underwound (negatively supercoiled) or overwound (positively supercoiled) DNA. These DNA topological changes regulate the interactions of DNA-binding proteins, including transcription factors, architectural proteins and topoisomerases. In order to advance our understanding of the structure and interactions of supercoiled DNA, we recently developed a single-molecule approach called Optical DNA Supercoiling (ODS). This method enables rapid generation of negatively supercoiled DNA (with between <5% and 70% lower helical twist than nonsupercoiled DNA) using a standard dual-trap optical tweezers instrument. ODS is advantageous as it allows for combined force spectroscopy, fluorescence imaging, and spatial control of the supercoiled substrate, which is difficult to achieve with most other approaches. Here, we describe how to generate negatively supercoiled DNA using dual-trap optical tweezers. To this end, we provide detailed instructions on the design and preparation of suitable DNA substrates, as well as a step-by-step guide for how to control and calibrate the supercoiling density produced.

Intraflagellar transport: Derailing causes turnarounds.

In intraflagellar transport, protein complexes travel along cilia from base to tip without stopping and change direction only at the tip. A new study shows that this directional change does not depend on any tip-associated machinery.

Mechanisms of Regulation in Intraflagellar Transport.

Cilia are eukaryotic organelles essential for movement, signaling or sensing. Primary cilia act as antennae to sense a cell's environment and are involved in a wide range of signaling pathways essential for development. Motile cilia drive cell locomotion or liquid flow around the cell. Proper functioning of both types of cilia requires a highly orchestrated bi-directional transport system, intraflagellar transport (IFT), which is driven by motor proteins, kinesin-2 and IFT dynein. In this review, we explore how IFT is regulated in cilia, focusing from three different perspectives on the issue. First, we reflect on how the motor track, the microtubule-based axoneme, affects IFT. Second, we focus on the motor proteins, considering the role motor action, cooperation and motor-train interaction plays in the regulation of IFT. Third, we discuss the role of kinases in the regulation of the motor proteins. Our goal is to provide mechanistic insights in IFT regulation in cilia and to suggest directions of future research.

Quantitative Acoustophoresis.

Studying cellular mechanics allows important insights into its cytoskeletal composition, developmental stage, and health. While many force spectroscopy assays exist that allow probing of mechanics of bioparticles, most of them require immobilization of and direct contact with the particle and can only measure a single particle at a time. Here, we introduce quantitative acoustophoresis (QAP) as a simple alternative that uses an acoustic standing wave field to directly determine cellular compressibility and density of many cells simultaneously in a contact-free manner. First, using polymeric spheres of different sizes and materials, we verify that our assay data follow the standard acoustic theory with great accuracy. We furthermore verify that our technique not only is able to measure compressibilities of living cells but can also sense an artificial cytoskeleton inside a biomimetic vesicle. We finally provide a thorough discussion about the expected accuracy our approach provides. To conclude, we show that compared to existing methods, our QAP assay provides a simple yet powerful alternative to study the mechanics of biological and biomimetic particles.

A local interplay between diffusion and intraflagellar transport distributes TRPV-channel OCR-2 along C. elegans chemosensory cilia.

To survive, Caenorhabditis elegans depends on sensing soluble chemicals with transmembrane proteins (TPs) in the cilia of its chemosensory neurons. Cilia rely on intraflagellar transport (IFT) to facilitate the distribution of cargo, such as TPs, along the ciliary axoneme. Here, we use fluorescence imaging of living worms and perform single-molecule tracking experiments to elucidate the dynamics underlying the ciliary distribution of the sensory TP OCR-2. Quantitative analysis reveals that the ciliary distribution of OCR-2 depends on an intricate interplay between transport modes that depends on the specific location in the cilium: in dendrite and transition zone, directed transport is predominant. Along the cilium motion is mostly due to normal diffusion together with a small fraction of directed transport, while at the ciliary tip subdiffusion dominates. These insights in the role of IFT and diffusion in ciliary dynamics contribute to a deeper understanding of ciliary signal transduction and chemosensing.

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.

Duplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex.

Topoisomerase IIIα is a type 1A topoisomerase that forms a complex with RMI1 and RMI2 called TRR in human cells. TRR plays an essential role in resolving DNA replication and recombination intermediates, often alongside the helicase BLM. While the TRR catalytic cycle is known to involve a protein-mediated single-stranded (ss)DNA gate, the detailed mechanism is not fully understood. Here, we probe the catalytic steps of TRR using optical tweezers and fluorescence microscopy. We demonstrate that TRR forms an open gate in ssDNA of 8.5 ± 3.8 nm, and directly visualize binding of a second ssDNA or double-stranded (ds)DNA molecule to the open TRR-ssDNA gate, followed by catenation in each case. Strikingly, dsDNA binding increases the gate size (by ~16%), while BLM alters the mechanical flexibility of the gate. These findings reveal an unexpected plasticity of the TRR-ssDNA gate size and suggest that TRR-mediated transfer of dsDNA may be more relevant in vivo than previously believed.

Direct imaging of intraflagellar-transport turnarounds reveals that motors detach, diffuse, and reattach to opposite-direction trains.

Intraflagellar transport (IFT), a bidirectional intracellular transport mechanism in cilia, relies on the cooperation of kinesin-2 and IFT-dynein motors. In Caenorhabditis elegans chemosensory cilia, motors undergo rapid turnarounds to effectively work together in driving IFT. Here, we push the envelope of fluorescence imaging to obtain insight into the underlying mechanism of motor turnarounds. We developed an alternating dual-color imaging system that allows simultaneous single-molecule imaging of kinesin-II turnarounds and ensemble imaging of IFT trains. This approach allowed direct visualization of motor detachment and reattachment during turnarounds and accordingly demonstrated that the turnarounds are actually single-motor switching between opposite-direction IFT trains rather than the behaviors of motors moving independently of IFT trains. We further improved the time resolution of single-motor imaging up to 30 ms to zoom into motor turnarounds, revealing diffusion during motor turnarounds, which unveils the mechanism of motor switching trains: detach-diffuse-attach. The subsequent single-molecule analysis of turnarounds unveiled location-dependent diffusion coefficients and diffusion times for both kinesin-2 and IFT-dynein motors. From correlating the diffusion times with IFT train frequencies, we estimated that kinesins tend to attach to the next train passing in the opposite direction. IFT-dynein, however, diffuses longer and lets one or two trains pass before attaching. This might be a direct consequence of the lower diffusion coefficient of the larger IFT-dynein. Our results provide important insights into how motors can cooperate to drive intracellular transport.

Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.

The combination of DNA force spectroscopy and polarization microscopy of fluorescent DNA intercalator dyes can provide valuable insights into the structure of DNA under tension. These techniques have previously been used to characterize S-DNA-an elongated DNA conformation that forms when DNA overstretches at forces ≥ 65 pN. In this way, it was deduced that the base pairs of S-DNA are highly inclined, relative to those in relaxed (B-form) DNA. However, it is unclear whether and how topological constraints on the DNA may influence the base-pair inclinations under tension. Here, we apply polarization microscopy to investigate the impact of DNA pulling geometry, torsional constraint, and negative supercoiling on the orientations of intercalated dyes during overstretching. In contrast to earlier predictions, the pulling geometry (namely, whether the DNA molecule is stretched via opposite strands or the same strand) is found to have little influence. However, torsional constraint leads to a substantial reduction in intercalator tilting in overstretched DNA, particularly in AT-rich sequences. Surprisingly, the extent of intercalator tilting is similarly reduced when the DNA molecule is negatively supercoiled up to a critical supercoiling density (corresponding to ∼70% reduction in the linking number). We attribute these observations to the presence of P-DNA (an overwound DNA conformation). Our results suggest that intercalated DNA preferentially flanks regions of P-DNA rather than those of S-DNA and also substantiate previous suggestions that P-DNA forms predominantly in AT-rich sequences.

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.