Topoisomerase enzymes are essential for the regulation of DNA topology. Human topoisomerase IIIα is a Type 1A topoisomerase that exists as a complex with RMI1 and RMI2, known as TRR. The TRR complex can unlink entwined DNA strands and is known to be important for resolving DNA replication and recombination intermediates. It has recently been proposed that TRR can also relax transient negatively supercoiled loops of DNA generated by the translocase PICH and that this activity may help to facilitate the resolution of ultrafine anaphase bridges (UFBs) between segregating sister chromatids. However, the mechanism by which TRR interacts with, and processes, negatively supercoiled DNA is not well understood. Here, we establish a single-molecule strategy to simultaneously measure real-time changes in supercoiling density and visualize the interactions of TRR with underwound DNA using a combination of optical tweezers and fluorescence imaging. We demonstrate that TRR relaxes highly negatively supercoiled DNA in a processive manner and that the timescale for relaxation is less than the expected lifetime of the negatively supercoiled loops generated by PICH. We also show that in the absence of free protein in solution, TRR remains bound to the DNA for long time periods after the torsional stress has been released. Our findings provide a mechanistic basis for how TRR can relax negative supercoils, consistent with its proposed role in UFB resolution. Moreover, our assay could also be widely applied to study the interactions of other families of topoisomerases with negatively supercoiled DNA.

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. Analysis of the mechanical properties of mitotic chromosomes is key for understanding the robustness of chromosomes during cell division. It is now shown that chromosomes are highly mechanically heterogeneous.

Abstract The integrity of the axoneme – the microtubule (MT)-based core of the cilium – and intraflagellar transport (IFT) are interdependent. The mechanisms determining axoneme structure and dynamics have remained largely unknown, especially in primary cilia with a more variable architecture and longer MT singlet parts. Using fluorescence imaging in the phasmid neurons of C. elegans , we here demonstrate that β-tubulin isotype TBB-4 diffuses through the dendrite and employs a combination of anterograde IFT and diffusion to reach the sites of incorporation in the steady-state axoneme. Disrupting tubulin’s ability to bind to the IFT significantly reduces its share in the axoneme. We suggest that, in phasmid cilia, a constant supply of tubulin by IFT is required for steady-state length maintenance, in order to elevate soluble tubulin concentration near the axonemal tips and to promote MT stability.

During mitotic cell division, pliable interphase chromatin is transformed into stiff mitotic chromosomes able to withstand the pushing and pulling forces of the mitotic spindle. How the cell establishes this chromosome stiffness and the cellular consequences if this stiffness is disrupted, is unclear. Condensin complexes drive many of the structural changes in mitotic chromosomes. Here, we combine rapid protein depletion of Condensins I and II with live cell imaging and mechanical characterization of purified mitotic chromosomes to probe their role in mitotic chromosome mechanics. We show that Condensin I, but not Condensin II, is required to establish chromosome stiffness and chromatin elasticity, and yet is not required for maintaining these properties after chromosome formation. Nevertheless, metaphase depletion of Condensin I still leads to severe sister centromere cohesion defects. We propose that the chromatin loop network established by Condensin I is locked in place by an additional 'crosslinking' factor.

The interface of chromosomes enables them to interact with the cell environment and is crucial for their mechanical stability during mitosis. Here, we use Atomic Force Microscopy (AFM) to probe the interface and local micromechanics of the highly condensed and complex chromatin network of native human mitotic chromosomes. Our AFM images provide detailed snapshots of chromatin loops and Sister-Chromatids Intertwines. A scaling analysis of these images reveals that the chromatin surface has fractal nature. AFM-based Force Spectroscopy and microrheology further show that chromosomes can resist severe deformations, elastically recovering their initial shape following two characteristic timescales. Localized indentations over the chromatids reveal that the spatially varying micromechanics of the chromatin network is largely governed by chromatin density. Together, our AFM investigation provides insights into the structure and local mechanics of mitotic chromosomes, offering a toolbox for further characterization of complex biological structures, such as chromosomes, down to the nanoscale.

The primary cilium is a signaling organelle that extends from many cell types to detect and relay extracellular signals. Beyond its signaling role, the cilium also produces cilia-derived extracellular vesicles (cEVs), although the mechanisms underlying their biogenesis and functions remain poorly understood. We characterized the cEV biogenesis in vivo using ciliated sensory neurons of C. elegans. In response to sensory cues, interruption of the intraflagellar transport (IFT) -a ciliary trafficking machinery carrying cargoes along the cilium- occurs together with ciliary membrane fission, resulting in the release of cEVs. Similarly, mutants disrupting IFT and ciliary receptor trafficking also enhance cEV production. To investigate how IFT influences the rate and location of cEV biogenesis, we selected a membrane marker that spans the entire length of the ciliary membrane independently of IFT. Single-molecule tracking demonstrates that the tetraspanin TSP-6 enters and diffuses within the cilia and does it independently of IFT. Lack of receptor retrieval or receptor entry in the cilium induces membrane budding from ciliary or periciliary membranes, respectively. Prior to fission, these membrane buds get enriched in TSP-6 as well as signaling receptors. Coupling receptor buildup with their export by cEVs provides a mechanism to preserve ciliary function and to modulate ciliary signaling.

When cells divide, the newly replicated sister chromatids must be segregated evenly to the daughter cells. During mitosis, mechanical force is applied by spindle microtubules in 2 ways: first by pushing on chromosome arms to promote chromosome congression to the cell equator in metaphase, and then by pulling on kinetochores to promote sister chromatid disjunction during anaphase. For segregation to proceed faithfully, the pliable interphase chromatin must be transformed into stiff mitotic chromosomes able to withstand these forces. However, it is unclear how the cell establishes chromosome stiffness and what the consequences are for dividing cells if this stiffness is disrupted. Many of the structural changes imposed on chromosomes in mitosis are driven by Condensin complexes, in conjunction with Topoisomerase IIα. Here, we have combined rapid protein depletion and live cell imaging with in-depth mechanical characterization of purified mitotic chromosomes to probe the roles of Condensins I and II in the establishment and maintenance of the mechanical strength of mitotic chromosomes. We show that Condensin I, but not Condensin II, is required to establish chromosome stiffness and chromatin elasticity, and yet ceases to be required for the maintenance of these properties once chromosome formation has been completed. Nevertheless, depletion of Condensin I from already formed chromosomes still impacts centromeric chromatin and leads to a loss of sister centromere cohesion. We propose that the extensive chromatin loop network established by Condensin I is locked in place by Topoisomerase IIα mediated DNA catenation. ### Competing Interest Statement G.J.L.W., E.J.G.P. and I.D.H. own shares of LUMICKS. G.J.L.W. and E.J.G.P. serve on the technical advisory board of LUMICKS. European UnionEuropean Union, https://ror.org/019w4f821, 665233, 859853, 883240 Novo Nordisk FoundationNovo Nordisk Foundation, , NNF18OC0034948 Deutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft, , WI 5434/1-1 Danish National Research FoundationDanish National Research Foundation, https://ror.org/00znyv691, DNRF115

Anterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A, and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in Caenorhabditis elegans chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport most likely on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results show that IFT-B is first incorporated into IFT trains, followed by IFT-A, and finally BBSomes, indicating that the assembly of IFT trains is a highly regulated, step-wise process.

The transition zone (TZ) is a selective barrier that maintains ciliary compartmentalization by controlling protein entry and exit. Cilia assembly requires the crossing of this barrier by intraflagellar transport (IFT) trains, scaffolded by IFT-A and IFT-B complexes, which move cargo bidirectionally using kinesin-2 and dynein-2 motors. In Caenorhabditis elegans , IFT-A loss abolishes retrograde transport, resulting in truncated cilia packed with IFT material. Here, we show that blocking TZ assembly prevents dynein-2 and IFT-B accumulation inside IFT-A-deficient cilia and partially rescues axoneme length. Single-particle imaging reveals that this rescue occurs without recovery of retrograde IFT. Instead, IFT particles exit cilia by passively diffusing through the disrupted TZ. Moreover, IFT-A/TZ double mutants shed ciliary extracellular vesicles (EVs) abnormally enriched in IFT components, providing a second clearance route. We conclude that TZ removal alters ciliary responses to retrograde transport defects, promoting diffusion and EV release to clear IFT machinery and facilitate axoneme extension. Highlights ### Competing Interest Statement The authors have declared no competing interest. Fundação para a Ciência e Tecnologia, https://ror.org/00snfqn58, 2023.12458.PEX, 2022.01955.PTDC, UID/06304/2023 and LA/P/0050/2020, CEECIND/01985/2018 and CEECINSTLA/00012/2022 and CEECIND/00333/2017, UI/BD/152865/2022 and 2023.01378.BD Dutch Research Council, NWO; Project no. OCENW.M20.063

Intraflagellar transport (IFT) coordinates the transport of cargo in cilia and is essential for ciliary function. CILK1 has been identified as a key regulator of IFT. The mechanism by which it acts has, however, remained unclear. In this study, we use fluorescence imaging and single-molecule tracking in the phasmid cilia of live Caenorhabditis elegans to study the effect of the CILK1 homologue DYF-5 on the dynamics of the IFT. We show that in the absence of DYF-5, IFT components accumulate at the ciliary tip. Kinesin-II is no longer restricted to the proximal segment of the cilium but is present throughout the cilium, while its velocity is different from that of OSM-3. The frequency of IFT trains is reduced and in particular retrograde trains were rarely observed. In the absence of DYF-5, retrograde transport is vastly reduced, resulting in the accumulation of IFT components at the tip and depletion at the base. The latter results in impeded anterograde train assembly, resulting in fewer trains with irregular composition. Our results show that DYF-5 plays a key role in regulating the turnarounds of IFT trains at the ciliary tip.

During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. How do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? Here, we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone-depletion and theory to show that chromosomal elasticity originates from the chromatin fiber behaving as a flexible polymer, whereas energy dissipation can be explained by interactions between chromatin loops. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.

Summary/Abstract Anterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in C. elegans chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results indicate that the assembly of IFT trains is a step-wise process involving the subsequent incorporation of first IFT-B, then IFT-A and finally BBSomes.

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.

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.

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.

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

surrounding protein. In particular, tight target binding to the 5’ seed region al- ters the kinetics of the 3’ non-seed interaction, resulting in kinetic heterogeneity in that interaction with the target mimic. This kinetic heterogeneity most likely arises from differential accessibility of the 3’ non-seed region of miRNA in Ago2, which adopts multiple conformations. In summary, our data indicate that both 5’ seed and 3’ non-seed-based target recognition is possible, but the accessibility and the kinetics of mRNA interactions is largely governed by the surrounding protein conformation as well as the guide-target complementarity.