In vitro Rekonstitution der Chromosomen-Separation mit Hilfe von optischen Pinzetten
Zusammenfassung der Projektergebnisse
When we look at the DNA of eukaryotic cells at different points in the cell cycle, it is striking how its appearance changes. Most of the time, in interphase, it is stored in the nucleus (e.g. the cell in the middle of the picture), different chromosomes are not discernable and it has a relatively dynamic conformation. But then, as the cell divides, the DNA is getting much denser and individual chromosomes can be identified (the left and right cells). This is necessary, since when the cell is dividing, there are substantial mechanical forces pulling the DNA into the new daughter cells. This motivated us to look deeply into the mechanical properties of mitotic chromosomes in order to understand how they can withstand these forces during mitosis. Using optical tweezers (highly focused laser beams that allow us to manipulate small objects), we developed a workflow to stretch individual mitotic chromosomes and characterize their response. We found that these chromosomes behaved very differently from what we expected: It did not behave like a single polymer, but like many different polymers put together. While this result was surprising to us, it makes sense, in that the chromosome is a very complex and heterogeneous hybrid material composed of many, many different components. We now showed, how this heterogeneity and complexity directly impacts chromosomal mechanics. Next, we set out to understand what causes these mechanical properties. We found two very different influences: On the one hand, the mechanical properties depended crucially on the ions present around the chromosome, with a particularly important role of physiological polyamine ions. These ions act as a sort of glue sticking neighboring DNA fibers together. If we take away this glue, chromosomes become large and fluffy, but more importantly, they are not able to withstand mechanical forces, which would have dramatic consequences in a living cell. On the other hand, from a molecular biology view point, we found that the protein complex condensin I (but surprisingly not its very similar brother condensin II) provides the chromosomes with stiffness. Condensins are the protein complexes that shape the DNA into its iconic form during cell division. Surprisingly, we found that it was not the presence of condensin I itself that mattered, but the structures it formed in the chromosome: Once it did its job, we could remove it without any impact on the chromosomal mechanics. In summary, in this project, we made a giant leap in our understanding of the mechanics of mitotic chromosomes. We described the mechanics in unprecedented detail and identified two main factors that define these mechanics: physiological ions and condensin I complexes.
Projektbezogene Publikationen (Auswahl)
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The mechanics of mitotic chromosomes. Quarterly Reviews of Biophysics, 54.
Man, T.; Witt, H.; Peterman, E. J. G. & Wuite, G. J. L.
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CENP-B-mediated DNA loops regulate activity and stability of human centromeres. Molecular Cell, 82(9), 1751-1767.e8.
Chardon, Florian; Japaridze, Aleksandre; Witt, Hannes; Velikovsky, Leonid; Chakraborty, Camellia; Wilhelm, Therese; Dumont, Marie; Yang, Wayne; Kikuti, Carlos; Gangnard, Stephane; Mace, Anne-Sophie; Wuite, Gijs; Dekker, Cees & Fachinetti, Daniele
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Nonlinear mechanics of human mitotic chromosomes. Nature, 605(7910), 545-550.
Meijering, Anna E. C.; Sarlós, Kata; Nielsen, Christian F.; Witt, Hannes; Harju, Janni; Kerklingh, Emma; Haasnoot, Guus H.; Bizard, Anna H.; Heller, Iddo; Broedersz, Chase P.; Liu, Ying; Peterman, Erwin J. G.; Hickson, Ian D. & Wuite, Gijs J. L.
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Ion-mediated condensation controls the mechanics of mitotic chromosomes.
Witt, Hannes; Harju, Janni; Chameau, Emma M.J.; Bruinsma, Charlotte M.A.; Clement, Tinka V.M.; Nielsen, Christian F.; Hickson, Ian D.; Peterman, Erwin J.G.; Broedersz, Chase P. & Wuite, Gijs J.L.
