Iddo Heller

Faithful chromosome segregation during mitosis relies on the formation of compact, individualized chromosomes that withstand drag and spindle-generated forces. Structural failure of mitotic chromosomes under force can disrupt the distribution of genetic material to daughter cells, causing aneuploidy or cancer. The overall mechanical properties of mitotic chromosomes have been suggested to arise from their structural heterogeneity. The magnitude and scale of this heterogeneity have not been measured, leaving its impact on chromosome mechanics unresolved. Here we show that chromosomes are highly mechanically heterogeneous: within one chromosome, the local stiffness can vary by up to two orders of magnitude. This extreme mechanical heterogeneity is exemplified by the centromere, which is an order of magnitude softer than the whole chromosome. These results demonstrate how the mechanical complexity of mitotic chromosomes gives rise to their emergent nonlinear mechanical behaviour, distinct from the polymer properties of their constituents. More broadly, we discuss how structural heterogeneity can shape the nonlinear responses of composite materials, with implications for both understanding biological assemblies and designing new synthetic materials.

Optical tweezers are a means to manipulate objects with light. With the technique, microscopically small objects can be held and steered, allowing for accurate measurement of the forces applied to these objects. Optical tweezers can typically obtain a nanometer spatial resolution, a picoNewton force resolution, and a millisecond time resolution, which makes the technique well suited for the study of biological processes from the single-cell down to the single-molecule level. In this chapter, we aim to provide an introduction to the use of optical tweezers for single-molecule analyses. We start from the basic principles and methodology involved in optical trapping, force calibration, and force measurements. Next, we describe the components of an optical tweezers setup and their experimental relevance. Finally, we will provide an overview of the broad applications in context of biological research, with the emphasis on the measurement modes, experimental assays, and possible combinations with fluorescence microscopy techniques. Key words Optical tweezers Optical trap Single molecule Fleezers Trap stiffness calibration Force spectroscopy Instrument design Molecular motors DNA-protein interactions

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. Key words Optical tweezers Thermal effects Heat generation Temperature control Trap calibration Temperature quantification

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. Key words Stimulated emission depletion microscopy (STED) Super-resolution fluorescence microscopy Dual-trap optical tweezers Focal-intensity distribution Aberration correction Alignment

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 TOP2A⁵specifically 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.

Self-assembling patchy colloidal particles form a promising platform to create designer soft materials. To dress such systems with mechanical functionality, one can take inspiration from biological structures such as the cell's cytoskeleton, which consists of semiflexible filaments, whose mechanical behavior give the cell its unique mechanical properties. Here we present mechanical experiments on analogs of biological fibers, semiflexible “colloidal polymers” made from bonded patchy colloidal particles. We use optical tweezers to probe their extreme mechanics under increasingly high compressions and we reveal a rich nonlinear mechanical response involving buckling, viscoelastic creep, and ultimately break-up. We characterize and model this response using elastic and viscoelastic models involving Euler buckling and stress relaxation. This allows us to identify the critical Euler buckling force, and relate the critical bending at break-up to the finite patch size of the colloids. These results demonstrate the crucial role of the patch-patch interactions in the mechanics of self-assembled colloidal materials, and they provide mechanical relationships that are essential to design functional colloidal architectures inspired by nature.

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.

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.

Optical tweezers and single-molecule polarization imaging reveal structural features of overstretched DNA.