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The propagation of life relies on the accurate replication and distribution of genomes. When sister chromatids segregate in anaphase, pieces of DNA that connect them are stretched into structures that are known as anaphase DNA bridges. These bridges contain catenated, incompletely replicated, or damaged DNA and their resolution before cytokinesis is important to prevent the transmission of instable genomes to the arising daughter cells. We want to understand at a molecular and mechanistic level how an ensemble of DNA helicases, translocases, and topoisomerases recognizes and processes DNA bridges in dividing human cells.
Image: a HeLa cell getting ready to divide (courtesy of Stefano Maffini).
Biochemical reconstitution is a powerful tool to study the protein machinery that orchestrates a process as intricate as a mitotic cell division. We reconstitute isolated parts of the protein-DNA machinery from purified components and study their properties. The ability to then systematically mix and modify constituents and regulators enables meaningful biochemistry, structural biology, and single-molecule biophysics. We test the implications that emerge from our in vitro experiments in human cell lines.
Pim studied Molecular Life Sciences in Nijmegen, the Netherlands. He moved to Vienna in 2009 to study sister chromatid cohesion in the lab of Jan-Michael Peters at the IMP (PhD). In 2015, he joined the lab of Andrea Musacchio (MPI-MOPH, Germany) to investigate how kinetochores attach to the mitotic spindle. In August 2023, Pim started as a group leader at the Max Perutz Labs.
We are happy with a small contribution to a very nice study by Colin Stok, Marcel van Vugt, and colleagues. The authors identified factors that become essential for viability in the absence of PICH (which processes anaphase DNA bridges). Among the 10 hits, there was 1 protein of unknown function that turns out to interact with FIGNL1 and RAD51 at replication forks. Alphafold predicted a very stable complex between FIGNL1 and FIRRM (check the pAE plot). Read the study here: [Stok et al., Cell Reports, 2023, https://doi.org/10.1016/j.celrep.2023.112668]
Ndc80 complexes are rod-like complexes that bind microtubules on one end and dock onto chromosomes at their other end. Using biochemistry, single-molecule biophysics, and cell biology, we discovered how a small sequence within the long coiled coil, known as the loop, promotes the formation of Ndc80 arrays on microtubules. This is important for chromosome congression during cell division, presumably because it allows for a cooperative attachment to the very end of a microtubule: [Polley et al., The EMBO Journal, 2023, https://doi.org/10.15252/embj.2022112504]
As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities and need to be recapitulated in vitro for the structural and functional characterization of mitotic protein machinery. In this paper, we describe how to get recombinant CDK1:Cyclin-B complexes that are efficiently phosphorylated at their T-loop, a prerequisite for kinase activity: [Huis in ’t Veld et al., Protein Science, 2022; https://doi.org/10.1002/pro.4233]
We engineered streptavidin scaffolds into an assembly with a single biotin binding site and up to nine covalently bound Ndc80 complexes. These spider-like objects span distances of well over a 100 nanometer. Collaborators at the TU Delft in the Netherlands attached these engineered Ndc80 multimers to the end of a homemade DNA origami nanospring to characterise their binding to dynamic microtubules: [Nick Maleki et al., Journal of Cell Science, 2022; https://doi.org/10.1242/jcs.260154]
FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics.
Stok, Colin; Tsaridou, Stavroula; van den Tempel, Nathalie; Everts, Marieke; Wierenga, Elles; Bakker, Femke J; Kok, Yannick; Alves, Inês Teles; Jae, Lucas T; Raas, Maximilian W D; Huis In 't Veld, Pim J; de Boer, H Rudolf; Bhattacharya, Arkajyoti; Karanika, Eleftheria; Warner, Harry; Chen, Mengting; van de Kooij, Bert; Dessapt, Julien; Ter Morsche, Lars; Perepelkina, Polina; Fradet-Turcotte, Amelie; Guryev, Victor; Tromer, Eelco C; Chan, Kok-Lung; Fehrmann, Rudolf S N; van Vugt, Marcel A T M
Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding
Polley, Soumitra; Müschenborn, Helen; Terbeck, Melina; Antoni, Anna De; Vetter, Ingrid R.; Dogterom, Marileen; Musacchio, Andrea; Volkov, Vladimir A.; Huis in t Veld, Pim J.
Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.
Huis In 't Veld, Pim J; Wohlgemuth, Sabine; Koerner, Carolin; Müller, Franziska; Janning, Petra; Musacchio, Andrea
Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling.
Huis In 't Veld, Pim J; Volkov, Vladimir A; Stender, Isabelle D; Musacchio, Andrea; Dogterom, Marileen
Voyage of the Starships: giant transposons as crucibles of evolution
Parthenogenesis, cryptobiosis, and the survival in extreme environmental conditions
Evading ageing: Mitochondrial and proteostatic adaptations in oocytes
Genomes in Rhodnius prolixus symbiotic system
Stem cells, immune evasion and metastasis in colorectal cancer
Ubiquitin & Friends Symposium 2024
The Ubiquitin & Friends Symposium is an annual international meeting taking place in the beautiful capital of Austria, aiming to bring together scholars from various fields studying ubiquitin/Ubl biology and protein degradation in a personal, family-like atmosphere, as suggested by the name.
The evolution and development of mollusc shells
Unraveling the Complexity of Crossover Regulation in C. elegans
Dynamics of 3D Genome Structure and Function
How superworms can help to solve our plastic waste crisis
Title to be announced
New players in an old pathway: biology of methanogens of the TACK superphylum
Shaping morphogen gradients: from molecules to tissues and back
Title to be announced
Studying stressed cells by in situ structural biology
Exploring Microbial Resilience: Unravelling Escherichia coliand#x27;s Stress Response at the Level of Protein Synthesis
Arbuscular mycorrhiza development and function
Deep homology and deep diversity: Evolving genetic toolkits for making and sensing light
The evolution of cell type identity and tissue microecology at the fetal-maternal interface
The unanticipated roles of PICIs and phages in bacterial evolution
Chemical targeting of subcellular protein localization
Origin and diversification of gut-derived organs in chordates
Job's Dilemma for the Genome: Why Bad Things Happen to Good Chromosomes
Connections between carbon and nitrogen cycling in the ocean
Understanding how the DNA-loop-extruding protein complex Condensin folds a chromatinized genome into mitotic chromosomes
DrugMap: A quantitative pan-cancer analysis of cysteine ligandability
From Roads to Rivers? Occurrence and environmental fate of tire and road wear particles and of tire-related chemicals
FENS 2024 Satellite event: Home cage behavior monitoring at the interface of animal welfare and neuroscience
Striking physiology and cell biology of (marine) environmental microorganisms
Mechanisms controlling maintenance of cohesin dependent loops
Title to be announced