Biology subjects

Huis in 't Veld, P. J.

Publications and source records attributed to Huis in 't Veld, P. J..

8 recordsLinked to original sources

bioRxiv2026

CIP2A tetramerization is required for mitotic DNA repair

DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.

cell biology↗

bioRxiv2025

Microtubule end stabilisation by cooperative oligomers of Ska and Ndc80 complexes

During mitosis, properly aligned chromosomes stabilise microtubule ends with the help of kinetochores to ensure timely segregation of chromosomes. Microtubule-binding components of the human outer kinetochore, such as Ndc80 and Ska complexes, are present in multiple copies and together bind several microtubule ends, creating a highly multivalent binding interface. Whereas Ndc80:Ndc80 and Ndc80:microtubule binding is crucial for interface stability, Ndc80 alone in absence of Ska is unable to support stable kinetochore-attachments. Using cryoET, we demonstrate that oligomeric Ndc80:Ska assemblies stabilise microtubule ends against shortening by strengthening lateral contacts between tubulin protofilaments at microtubule plus-ends. We further identify a point mutation within the SKA1 microtubule-binding domain that does not affect microtubule-binding of individual Ska molecules, but does abolish Ska:Ska interactions. Finally, we report that oligomerisation of Ska, in a cooperative fashion together with the Ndc80, is necessary to maintain stable microtubule attachments both in vivo and in vitro.

biochemistry↗

bioRxiv2022

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence identified across eukaryotes and predicted to interrupt the Ndc80 coiled-coil, promotes direct interactions between full-length Ndc80 complexes. Both in dividing cells and in a fully reconstituted system, these interactions are essential for the formation of force-resistant kinetochore-microtubule attachments, explaining why deletion of the loop ablates chromosome congression. The loop may fold into a more rigid structure than previously assumed and we identify point mutations that impair Ndc80-Ndc80 interactions and recapitulate the effects of loop depletion. The loop also promotes spindle checkpoint signaling, suggesting that the organisation of adjacent Ndc80 complexes is crucial to couple kinetochore-microtubule binding to cell cycle control.

biochemistry↗

bioRxiv2022

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.

biophysics↗

bioRxiv2022

Strain stiffening of the Ndc80 complex

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 (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, which 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. SIGNIFICANCEBy time-tracing the stiffness of microtubule end-tracking Ndc80 complexes in optical trap experiments, we detect strain stiffening, and, thereby, provide new insights into the elastic properties of the Ndc80 complex. The strain stiffening is robust against mutations in the Ndc80 complex. We relate strain stiffening to the structure of the Ndc80 complex by means of a simple polymer model, to thermal fluctuations of the microtubule, and to the flexibility of force-generating flaring protofilaments at the tip of the microtubule. Since Ndc80 complexes play a major role for transmitting force from microtubule ends to the kinetochore, their elastic properties are of great interest for a deeper understanding of chromosome dynamics in the mitotic spindle.

biophysics↗

bioRxiv2021

Structure of the human FERRY Rab5 effector complex

Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction leads to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, where two arm-like appendages of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. The coiled-coil domains of Fy-2 are flexible and project into opposite directions from the core. While the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. The FERRY structure provides novel mechanistic insights into long-distance mRNA transport.

biochemistry↗

bioRxiv2019

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. Under force, Ska stabilizes end-on microtubule attachments in parallel with the Ndc80-tail, which we reveal to be essential for end-tracking by Ndc80 multimers. 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.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC=\"FIGDIR/small/675363v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (38K):\norg.highwire.dtl.DTLVardef@9e6207org.highwire.dtl.DTLVardef@35f668org.highwire.dtl.DTLVardef@c43242org.highwire.dtl.DTLVardef@bfcc13_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2017

NDC80 clustering modulates microtubule dynamics under force

Multivalency, the presence of multiple interfaces for intermolecular interactions, underlies many biological phenomena, including receptor clustering and cytosolic condensation. One of its ultimate purposes is to increase binding affinity, but systematic analyses of its role in complex biological assemblies have been rare. Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved but poorly characterized feature of kinetochores, the points of attachment of chromosomes to spindle microtubules. To address its significance, we engineered modules allowing incremental addition of NDC80 complexes. The modules residence time on microtubules increased exponentially with the number of NDC80 complexes. While modules containing a single NDC80 complex were unable to track depolymerizing microtubules, modules with two or more complexes tracked depolymerizing microtubules and stiffened the connection with microtubules under force. Cargo-conjugated modules of divalent or trivalent NDC80 stalled and rescued microtubule depolymerization in a force-dependent manner. Thus, multivalent microtubule binding through NDC80 clustering is crucial for force-induced modulation of kinetochore-microtubule attachments.

biophysics↗