Periodic Reporting for period 2 - MONOCHROME (Disentangling metaphase chromosome organisation one chromosome at a time)
Reporting period: 2022-07-01 to 2023-12-31
Chromosomes assume their most compact state during metaphase just before they are separated. In this process of cell division the chromosomes experience high forces and genomic defects can occur then. Many techniques have built considerable understanding of metaphase chromosome structure and a multitude of models have been put forward how cells organize their chromosomes during metaphase. Yet, given the complexity of the process and limitations of the methods to study them, it is far from being fully understood. The breakthrough opportunity in this regard is the development of tools that allow real-time, 3D, super-resolution imaging and manipulation of entire non-fixed metaphase chromosomes.
Here we propose to quantitatively image the proteins that establish the architecture of metaphase chromosomes and disentangle the connection between its architecture, internal protein dynamics and mechanics at the multi-protein as well as the single-molecule level. For this project we plan to expand the combination of optical manipulation and fluorescent microscopy by introducing force-induced expansion microscopy together with advanced labeling and imaging techniques that ultimately will permit real-time, 3D, super-resolution quantitative analysis of complex (protein) structures within native non-fixed metaphase chromosomes. With this kind of instrument it becomes possible to validate and/or challenge the current models of metaphase organization as well as explore the physical properties of chromosomes but also study chromosome separation dynamics. Finally, we can directly verify the models that exist explaining chromosome organization in either healthy or diseased cells.
We are already succeeding to bring this dream a significant step closer, by combining optical manipulation with powerful fluorescent techniques. The new instruments that we have developed represents the state of the art and is able to make real-time movies of many proteins binding, handling and moving about on DNA. Most significantly the microscope can do this at high protein concentrations on and around the DNA which is exactly like the situation inside cells. This combination of single-molecule manipulation and fluorescence has an enormous potential for the quantification of biological systems. Moreover, the instrument is very flexible in how it can be used and therefore it permits the study of any kind of DNA-proteins. We have patented this technology which, in turn, enabled us to start commercializing it in a spin-off company (LUMICKS) which now makes this new microscope accessible for research labs around the world.
Next, by boosting our understanding of disease on a molecular level we open up the potential to translate it into diagnostic or treatment options. In MONOCHROME, we open-up the quantitative, molecular insight of met-aphase chromosome structure and mechanics. This is important because problems in the process of segregating chromosomes can have severe effects, as the daughter cells might end up with the wrong amount (aneuploidy) and/or damaged chromosomes. Such chromosomal abnormalities are characteristic of many disorders such as cancer and neurological disorders such as fragile X syndrome (FXS). Quantitative methods of investigating non-fixed metaphase chromosomes might help shed light on such abnormalities because of the unparalleled control and imaging resolution we will have for investigating chromosomes.
Here we propose to quantitatively image the proteins that establish the architecture of metaphase chromosomes and disentangle the connection between its architecture, internal protein dynamics and mechanics at the multi-protein as well as the single-molecule level. For this project we plan to expand the combination of optical manipulation and fluorescent microscopy by introducing force-induced expansion microscopy together with advanced labeling and imaging techniques that ultimately will permit real-time, 3D, super-resolution quantitative analysis of complex (protein) structures within native non-fixed metaphase chromosomes. With this kind of instrument it becomes possible to validate and/or challenge the current models of metaphase organization as well as explore the physical properties of chromosomes but also study chromosome separation dynamics. Finally, we can directly verify the models that exist explaining chromosome organization in either healthy or diseased cells.
We are already succeeding to bring this dream a significant step closer, by combining optical manipulation with powerful fluorescent techniques. The new instruments that we have developed represents the state of the art and is able to make real-time movies of many proteins binding, handling and moving about on DNA. Most significantly the microscope can do this at high protein concentrations on and around the DNA which is exactly like the situation inside cells. This combination of single-molecule manipulation and fluorescence has an enormous potential for the quantification of biological systems. Moreover, the instrument is very flexible in how it can be used and therefore it permits the study of any kind of DNA-proteins. We have patented this technology which, in turn, enabled us to start commercializing it in a spin-off company (LUMICKS) which now makes this new microscope accessible for research labs around the world.
Next, by boosting our understanding of disease on a molecular level we open up the potential to translate it into diagnostic or treatment options. In MONOCHROME, we open-up the quantitative, molecular insight of met-aphase chromosome structure and mechanics. This is important because problems in the process of segregating chromosomes can have severe effects, as the daughter cells might end up with the wrong amount (aneuploidy) and/or damaged chromosomes. Such chromosomal abnormalities are characteristic of many disorders such as cancer and neurological disorders such as fragile X syndrome (FXS). Quantitative methods of investigating non-fixed metaphase chromosomes might help shed light on such abnormalities because of the unparalleled control and imaging resolution we will have for investigating chromosomes.
In the first half of the project we made important progress in three areas as summarized below.
1) In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped metaphase chromosomes. This dramatic metamorphosis is driven mainly by the combined action of condensins and Topoisomerase IIα (TOP2A) and has been observed using microscopy for over a century. Nevertheless, remarkably little is known about the structural organization of a mitotic chromosome. In this first and the most essential project, we developed a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behavior, distinct from classical polymer models. To explain this anomalous stiffening, we introduce a Hierarchical Worm-like Chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A specifically in mitosis, we provide evidence of a role for TOP2A in the preservation of chromosome compaction. Our innovations open the door to a wide array of new investigations on the structure and dynamics of both healthy and disease-associated chromosomes.
2) In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. In this second project, we present an in vitro single-molecule assay that mimicks chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.
3) During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. In this third project we wondered how do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? To address this we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone-depletion and theory to show that chromosomal elasticity originates from the chromatin fiber behaving as a flexible polymer, whereas energy dissipation can be explained by interactions between chromatin loops. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.
1) In preparation for mitotic cell division, the nuclear DNA of human cells is compacted into individualized, X-shaped metaphase chromosomes. This dramatic metamorphosis is driven mainly by the combined action of condensins and Topoisomerase IIα (TOP2A) and has been observed using microscopy for over a century. Nevertheless, remarkably little is known about the structural organization of a mitotic chromosome. In this first and the most essential project, we developed a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native chromosomes to be conducted under tightly controlled experimental conditions. We have used this method to extensively characterize chromosome mechanics and structure. Notably, we find that under increasing mechanical load, chromosomes exhibit nonlinear stiffening behavior, distinct from classical polymer models. To explain this anomalous stiffening, we introduce a Hierarchical Worm-like Chain model that describes the chromosome as a heterogeneous assembly of nonlinear worm-like chains. Moreover, through inducible degradation of TOP2A specifically in mitosis, we provide evidence of a role for TOP2A in the preservation of chromosome compaction. Our innovations open the door to a wide array of new investigations on the structure and dynamics of both healthy and disease-associated chromosomes.
2) In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. In this second project, we present an in vitro single-molecule assay that mimicks chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.
3) During mitosis in eukaryotic cells, mechanical forces generated by the mitotic spindle pull the sister chromatids into the nascent daughter cells. In this third project we wondered how do mitotic chromosomes achieve the necessary mechanical stiffness and stability to maintain their integrity under these forces? To address this we use optical tweezers to show that ions involved in physiological chromosome condensation are crucial for chromosomal stability, stiffness and viscous dissipation. We combine these experiments with high-salt histone-depletion and theory to show that chromosomal elasticity originates from the chromatin fiber behaving as a flexible polymer, whereas energy dissipation can be explained by interactions between chromatin loops. Taken together, we show how collective properties of mitotic chromosomes, a biomaterial of incredible complexity, emerge from molecular properties, and how they are controlled by the physico-chemical environment.
The workflow that we developed described in (1) above represent a true breakthrough with respect to what has been possible so far for the investigation of whole mitotic chromosomes.
It also enables us to push forward with the project as described in our proposal which includes research on the protein scaffold of chromosomes, the unravelling of the chromosomes as well as the controlled segregation of the sister chromatids.
It also enables us to push forward with the project as described in our proposal which includes research on the protein scaffold of chromosomes, the unravelling of the chromosomes as well as the controlled segregation of the sister chromatids.