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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.
Dr. Bohr-Gasse 9
1030 Vienna
Room: 5.515
+43-1-4277-74830
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]
Victor Cheng-chieh Chao
PhD Student +43-1-4277-74831
Room: 5.515
Pim Huis in 't Veld
Group Leader +43-1-4277-74830
Room: 5.515
Strain stiffening of Ndc80 complexes attached to microtubule plus ends
F Schwietert, VA Volkov, PJ Huis in 't Veld, M Dogterom, A Musacchio, ...
Biophysical Journal
Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding
S Polley, H Müschenborn, M Terbeck, A De Antoni, IR Vetter, M Dogterom, ...
bioRxiv 505310 (doi.org/10.1101/2022.08.25.505310)
The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex
C Chen, V Piano, A Alex, S Han, PJ Huis in 't Veld, B Roy, A Musacchio, ...
bioRxiv 498198 (https://doi.org/10.1101/2022.06.29.49819)
Estimation of microtubule-generated forces using a DNA origami nanospring
A Nick Maleki, PJ Huis in 't Veld, A Akhmanova, M Dogterom, VA Volkov
Journal of Cell Science 136 (5), jcs260154
Reconstitution and use of highly active human CDK1: Cyclin‐B: CKS1 complexes
PJ Huis in 't Veld, S Wohlgemuth, C Koerner, F Müller, P Janning, ...
Protein Science
Structure of the human FERRY Rab5 effector complex
D Quentin, JS Schuhmacher, BU Klink, J Lauer, TR Shaikh, ...
bioRxiv 449265 (doi.org/10.1101/2021.06.21.449265)
CDC20 assists its catalytic incorporation in the mitotic checkpoint complex
V Piano, A Alex, P Stege, S Maffini, GA Stoppiello, PJ Huis in ’t Veld, ...
Science 371 (6524), 67-71
Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint
LA Allan, MC Reis, G Ciossani, PJ Huis in ’t Veld, S Wohlgemuth, ...
The EMBO Journal
PJ Huis in ’t Veld, VA Volkov, ID Stender, A Musacchio, M Dogterom
eLife 8, e49539
G Ciossani, K Overlack, A Petrovic, PJ Huis in ’t Veld, C Koerner, ...
J. Biol. Chem. 293 (26), 10084-10101
V Volkov, PJ Huis in ’t Veld, M Dogterom, A Musacchio
eLife 7, e36764
Molecular basis of outer kinetochore assembly on CENP-T
PJ Huis in ’t Veld, S Jeganathan, A Petrovic, P Singh, J John, V Krenn, ...
eLife 5, e21007
Cohesin acetylation and Wapl‐Pds5 oppositely regulate translocation of cohesin along DNA
M Kanke, E Tahara, PJ Huis in ’t Veld, T Nishiyama
EMBO journal 35, 2686 - 2698
Rapid movement and transcriptional re‐localization of human cohesin on DNA
IF Davidson, D Goetz, MP Zaczek, MI Molodtsov, PJ Huis in ’t Veld, ...
EMBO Journal 35, 2671-2685
Topology and structure of an engineered human cohesin complex bound to Pds5B
MT Hons, PJ Huis in ’t Veld, J Kaesler, P Rombaut, A Schleiffer, F Herzog, ...
Nature Communications 7, 12523
biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes
F Weissmann, G Petzold, R VanderLinden, PJ Huis in ’t Veld, NG Brown, ...
Proceedings of the National Academy of Sciences 113 (19), E2564 - E2569
A Friese, AC Faesen, PJ Huis in ’t Veld, J Fischböck, D Prumbaum, ...
Nature Communications 7, 11407
Characterization of a DNA exit gate in the human cohesin ring
PJ Huis in ’t Veld, F Herzog, R Ladurner, IF Davidson, S Piric, E Kreidl, ...
Science 346 (6212), 968-972
Cohesin’s ATPase activity couples cohesin loading onto DNA with Smc3 acetylation
R Ladurner, V Bhaskara, PJ Huis in ’t Veld, IF Davidson, E Kreidl, ...
Current Biology 24 (19), 2228-2237
J Medvedovic, A Ebert, H Tagoh, IM Tamir, TA Schwickert, M Novatchkova, ...
Immunity 39 (2), 229-244
T Nishiyama, MM Sykora, PJ Huis in ’t Veld, K Mechtler, JM Peters
Proceedings of the National Academy of Sciences 110 (33), 13404-13409
Reconstitution of the human cohesin complex
PJ Huis in ’t Veld
University of Vienna - othes.univie.ac.at/25712
JP Alao, PJ Huis in ’t Veld, F Buhse, P Sunnerhagen
Molecular microbiology 77 (1), 143-157
From chromosome folding by loop extrusion to anti-plasmid nucleases in bacteria
11:00 - 12:00
IMBA/GMI Lecture HallIdentifying and exploiting cell-state dependent metabolic programs
11:00 - 12:00 IMP Lecture HallMind matters. VBC mental health awareness
13:30 - 14:30 IMP Lecture HallChromosomes as Mechanical Objects: from E.coli to Meiosis to Mammalian cells
11:00 - 12:00 IMBA/GMI Lecture HallConvergent evolution of CO2-fixing liquid-liquid phase separation
11:30 - 12:30 GMI Orange Seminar RoomViral envelope engineering for cell type specific delivery
16:00 - 17:00 IMP Seminar Room 1.014/16New ways of leading: inclusive leadership and revising academic hierarchies
14:15 - 15:15 IMP Lecture HallTitle to be announced
14:00 - 15:00 Lecture Hall AandB, VBC5Decoding Molecular Plasticity in the Dark Proteome of the Nuclear Pore Complex
11:00 - 12:00 IMBA/GMI Lecture HallNeurodiversity in academia
14:00 - 15:00 IMP Lecture HallGene expression dynamics during the awakening of the zygotic genome
11:00 - 12:00 IMBA/GMI Lecture HallTitle to be announced
11:00 - 12:00