Rapid movement and transcriptional re-localization of human cohesin on DNA

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA + ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability. </p

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability

Estimation of microtubule-generated forces using a DNA origami nanospring

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation

Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly.BN/Marileen Dogterom LabBN/Bionanoscienc

Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin

Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP: Astrin heterodimer localizes to spindle microtubules and to mature microtubule-kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore-microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.BN/Marileen Dogterom LabBN/Bionanoscienc

Estimation of microtubule-generated forces using a DNA origami nanospring

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BN/Marileen Dogterom La

Estimation of microtubule-generated forces using a DNA origami nanospring

Microtubules are dynamic cytoskeletal filaments that can generate forces when polymerizing and depolymerizing. Proteins that follow growing or shortening microtubule ends and couple forces to cargo movement are important for a wide range of cellular processes. Quantifying these forces and the composition of protein complexes at dynamic microtubule ends is challenging and requires sophisticated instrumentation. Here, we present an experimental approach to estimate microtubule-generated forces through the extension of a fluorescent spring-shaped DNA origami molecule. Optical readout of the spring extension enables recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. DNA nanosprings enable multiplexing of force measurements and only require a fluorescence microscope and basic laboratory equipment. We validate the performance of DNA nanosprings against results obtained using optical trapping. Finally, we demonstrate the use of the nanospring to study proteins that couple microtubule growth and shortening to force generation

Strain stiffening of Ndc80 complexes attached to microtubule plus ends

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants (calponin homology (CH) domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.BN/Marileen Dogterom La