Rezultaty
The core of the research in the later phases of the project will be conducted on biobank materials and cell lines. We will submit the ethics approval and required documents for working with this material within this milestone. The consortium will decide before month 6 if TRACERX clinical trail samples are used or not. If they are used we will gather all the required documentation such as the informed consent and information sheets. Moreover, we will explicitly confirm to REA that appropriate consent has been given by the participants of TRACERX to allow their samples and data to be used in the present research. The confirmation and the required materials in this case will be submitted before month 24. Next, if TRACERX material will be used we will have it anonymized so there will not be personal sensitive data. The same applies for a policy for the handling of any incidental findings that might arise when studying samples from participants. By anonymizing the data incidental findings will not be tracable to a person.
Reproducible force-distance curvesReproducible force-distance curves that can be correlated with super-resolution images In this task we use the approaches developed in task 3.2 to enhance our understanding of the mechanical and structural properties of chromosomes by combining micro-mechanical manipulation with super-resolution imaging. The VU, UCPH and UCL will investigate chromosomes with known defects, such as chromosome fragility. The experiments will yield information in the differences between the structural and mechanical properties of healthy and diseased chromosomes. Our aim is to reveal correlations between protein structures and the mechanical response of chromosomes to elucidate the nature/origin of chromosome architecture as well as defects.
Real-time data of UFB formation and resolutionReal-time data of UFB formation and resolution The VU and UCPH groups will investigate whether UFBs can be visualised in whole metaphase chromosomes following the application of force to both sister chromatids by our optical tweezers. UFBs will be visualised using purified PICH-GFP protein. A question we aim to answer is whether PICH can act on its own or requires co-factors to access UFBs. This task will demonstrate our ability to access specific processes that take place on chromosomes and therefore acts as a roadmap for further fundamental science questions (as for exampled explored in task 1.6) that can be pursued with the instrument.
Detailed communication and dissemination planDetailed communication and dissemination plan and annual updates. CHROMAVISION will developed a stakeholder engagement analysis (see also table in section 2.2. of the proposal). This concept plan provides the starting point for the dissemination and exploitation strategy. Understanding stakeholder motivations will enable the consortium to effectively engage, communicate with and promote future dialogue between different stakeholders and will help with more effective and targeted communication strategies for the different groups. All results of CHROMAVISION selected for publication will preferentially be made available through Open Access (OA) publication for broad dissemination. Either Green or Gold OA publishing models will be used. Annual updates take place at month 12,24,36,48
Protocol for producing 3D super-resolution imagesProtocol for producing 3D super-resolution images of whole chromosomes The focus here is to bring imaging of metaphase chromosomes to a new level of resolution and control. The condensed chromosomes will be flown in our microfluidic chamber (WP2) and captured out of solution using the specific chromosome-microsphere handles developed in task 3.1. We will apply different imaging approaches, starting with confocal fluorescence microscope and 2D-STED approaches while holding a chromosome with two optically trapped microspheres, and ultimately aiming for 3D-STED imaging of chromosomes held with four microspheres, using the newly developed instrument of task 1.2. We envision that this super-resolution imaging of different kinds provided by UCPH and UCL will result in deep insights in chromosome structure.
LOC for single cell capture and lysis DTU will develop an opto-fluidic chip able to trap, visualise and lyse individual cells and separate metaphase chromosomes from cell lysate. The chip represents the other upstream element of the integrated device that is going to deliver chromosomes to the CTFM-3DSR instrument (see task 2.4). The microfluidic element for sample preparation – single cell capture, visualisation, lysing and chromosome sample cleaning – will be developed with three approaches in parallel: (1) Single cells are captured in parallel flow constrictions; (2) in 2-beam optical traps (stretchers), integrated on the plastic chip; (3) in a hydrogel turned on and off by a near-infrared laser. The captured cells are then visualized and lysed by buffer exchange. This will lead to a decision point at M24, which approach to use in the further developments. UCPH will use the devices as standalone tools for biological investigations (tasks 2.5).
LOC for integration with optical tweezers & STEDLOC for integration with optical tweezers & STED In this task we will apply the production ready method for producing a LOC device that is compatible with 3DSR-CTFM (WP1). Tests will be run to investigate the compatibility of the polymer devices with optical trapping and STED imaging and decide on the format for the LOC. The 3 possibilities are in decreasing preference order: all polymer device, hybrid glass-polymer device or a modular device. We will work on optimisation of the chips for the various tasks below. Eventually, this task will lead to integration of many optical components into the LOC device.
Instrument and workflow for chromosome studiesWorkflow for chromosome extraction, preparation, imaging and manipulation in cell-like environments. To perform and supervise ongoing proof-of-concept experiments to test the experimental instruments for chromosome purification, manipulation and visualisation.
Bead-handlesBead-handles that can be easily connected to chromosome ends or centromers This task aims to develop a protocol that allows the attachment of optically trapped microspheres to a metaphase chromosome. First, the VU will explore the use of microspheres coated with antibodies. To this end we will use antibodies derived by UCPH against telomere-binding proteins or antibodies against centromere factors. A completely different approach we will pursue involve PNA or RNA linkers. These linkers hybridise more strongly with a complementary DNA strand than DNA itself. Another approach involves aptamers, nucleic acids that specifically bind to ligands. The expectation is that within 6 months chromosomes can be handled by specific attachment of optically trapped microspheres to chromosomes.
LoC prototypes for single cell chromosome analysisProduction ready prototypes of plastic LOC for chromosome analysis starting from single cells DTU will establish the process chain and deliver prototypes of plastic LOC devices for opto-fluidic sensing and actuation of individual cells and chromosomes, to be integrated in the 3DSR-CTFM connected to the WP2 deliverables. The task comprises of (1) design of a 'master' for inject ion moulding; (2) production of polymeric devices using injection moulding & nano-imprinting; (3) post-processing by polymer-to-polymer bonding to seal the devices.
Second 3DSR-CTFM instrument3DSR-CTFM instrument that can be shipped to UCPH & UCL. To deliver by M36 an instrument that is robust, user-friendly and accessible enough to be operated by users in an biological or clinical research setting. This 2nd instrument will be moved to UCPH & UCL for rigorous use there in a biomedical research environment (task 1.6). This task integrates all of the developments of task 1.1-4 including the input obtained from WP2 & 3.
2 LOC to emulate chromatid segregation2 LOC to emulate chromatid segregation A micro-fluidic platform will be developed by DTU to emulate chromatid segregation, combining stagnant fluid volumes, fluorescence imaging, reagent/buffer exchange by diffusion, and thermophoretic forces via opto-thermal actuation. The chip represents an upstream element of the integrated device that is going to deliver chromosomes to the CTFM-3DSR instrument (see task 2.4). The chip will also function as a standalone device that can be used to investigate condensed chromosomes. DTU will develop and validate the chip performance on model samples. UCPH will validate them with real chromosome.
LOC for single cell chromosome analysisLOC for single cell chromosome analysis This task represents the integration of tasks 2.1-3 into a single opto-fluidic chip able to deliver all chromosomes from a single cell to on-chip chromosome analysis. The final goal is to integrate the cell lysis with the chromosome arraying of all chromosomes from a single cell that are isolated and cleaned for further on-chip analysis in a “pick-and-place” protocol, using optical tweezers. The chip integrated two-beam optical traps and hydrogel traps developed in task 2.3 will be further developed to generate a mesh of random traps – a ‘mine field’ - where the metaphase chromosomes can be arrayed and further processed before they are ‘delivered’ to the CTFM-3DSR instrument, as will be tested by VU and LUM. UCPH and VU will validate the chips with relevant samples on the integrated instrument in tasks 1.6 and 3.3.
Validation CTFM-SR3D in biological/medical settingValidation CTFM-SR3D in biological/medical setting. UCPH and UCL scientists will be trained to work with the second CTFM-SR3D instrument, first at LUMICKS and later at their institutes to carry-out the following experiments (in order of complexity): (1) Isolate meiotic chromosomes from normal and disease conditions to analyse the structure of recombination–driven paired homologous chromosomes. Can we discover why women over 35 years have an exponential increase in chromosome nondisjunction leading to aneuploidy in ova? (2) Develop tools for diagnosis of ‘chromosome diseases’ such as cancer/neurological disorders. This might entail generation of probes/paints that specifically recognise each chromosome to study the formation of chromosome translocations, chromosome fusions and aneuploidy in an automated way. This could develop into high throughput karyotyping in the future, a currently difficult and laborious procedure (3) Can we reconstitute the ‘search for homology’ between 2 recombining sequences placed on different chromosomes or between 2 homologous chromosomes?
Field tested LOC for metaphase chromosome analysisField tested LOC for metaphase chromosome analysis UCPH will validate the chips developed in task 2.2 and 2.3 with relevant samples and bio-chemistry in order to develop methods and techniques to use the chips. Results from these experiments will be used for further improvements of the chip technology. Once we are able to handle the biological samples robustly in the chips we will start testing them on biologically relevant questions such as chromosome segregation dynamics. In this task we would aim to use purified proteins to address whether sister chromatid disjunction can be triggered solely by a combination of Separase and Topoisomerase II. We will also investigate the use of a modified version of cohesin incorporating a TEV protease recognition site into the SCC1 subunit. Using purified TEV protease, we could then trigger dissolution of cohesion ring and hence destroy sister chromatid cohesion. Our recent unpublished data indicate that the PICH protein strongly stimulates the catalytic activity of Topo II. Hence, we will investigate the possible role of PICH in stimulating centromeric DNA decatenation. The results of this study will be used in task 3.4 which aims at high resolution real-time imaging of the process.
First working 3DSR-CTFM instrumentFirst working 3DSR-CTFM instrument suitable for VU/VUmc partner. Develop and construct 3DSR-CTFM system with 3D STED (M1-M24): To allow for super-resolution chromosome imaging, VU scientists will extend the capabilities of their 1D-STED-equipped CTFM instrument by including a spatial light modulator. This will tremendously enhance capabilities (1D to 3D) and performance of the instrument. The optical design will be further developed for implementation in LUM's instruments, converted into a mechanically feasible and stable CAD/CAM design and a prototype will be manufactured. In this 1st prototype instrument improvements of task 1.1 will be implemented.
Super-resolutions movies of centromeric protein dynamics of chromosome segregation Here, the VU and UCPH groups are planning to trigger and follow the dynamics of a segregating chromosome. These experiments build on the experience from tasks 2.2 & 2.5. The result of this task will be highly detailed spatial and temporal observations of the events taking place during the first steps of chromosome segregation. Such data is far beyond the current state of the art and would allow a biological understanding of segregation currently unthinkable.
Collaborative and public websitePublic website Set up an website to provide information to the wide public
Dissemination material & updatesDissemination material & updates By M9 we have the infrastructure ready for: • Website: the CHROMAVISION website serves to feed all relevant stakeholders, including the general public, with information on project progress. The PIs will be asked to post regular new items about their work. The PIs will also introduce the WPs in a two-minute introduction video that is posted online. The website will integrate social media. • Animations: CHROMAVISION is a complex project. In WP4 an animation video will be developed that is comprehensible for all relevant stakeholders. The animation video will be used in external presentations. • Newsletter: All stakeholders will be informed with a quarterly newsletter (open for subscription) in which the project progress and relevant updates from outside the consortium are presented to all stakeholders. • Social-media platforms will be used to create a general awareness of CHROMAVISION.
Publikacje
Autorzy:
Oren Lotan, Jonathan Bar-David, Cameron L.C. Smith, Sharon Yagur-Kroll, Shimshon Belkin, Anders Kristensen, Uriel Levy
Opublikowane w:
Nano Letters, Numer 17/9, 2017, Strona(/y) 5481-5488, ISSN 1530-6984
Wydawca:
American Chemical Society
DOI:
10.1021/acs.nanolett.7b02132
Autorzy:
Johan Eriksen, Brian Bilenberg, Anders Kristensen, Rodolphe Marie
Opublikowane w:
Review of Scientific Instruments, Numer 88/4, 2017, Strona(/y) 045101, ISSN 0034-6748
Wydawca:
American Institute of Physics
DOI:
10.1063/1.4979164
Autorzy:
Cameron L. C. Smith, Anil H. Thilsted, Jonas N. Pedersen, Tomas H. Youngman, Julia C. Dyrnum, Nicolai A. Michaelsen, Rodolphe Marie, Anders Kristensen
Opublikowane w:
ACS Nano, Numer 11/5, 2017, Strona(/y) 4553-4563, ISSN 1936-0851
Wydawca:
American Chemical Society
DOI:
10.1021/acsnano.6b08563
Autorzy:
Johan Eriksen, Julien Schira, Nadine Vincent, Celine Sabatel, Anders Kristensen, Rodolphe Marie
Opublikowane w:
Microelectronic Engineering, Numer 187-188, 2018, Strona(/y) 14-20, ISSN 0167-9317
Wydawca:
Elsevier BV
DOI:
10.1016/j.mee.2017.11.011
Autorzy:
Xiaolong Zhu, Wei Yan, Uriel Levy, N. Asger Mortensen, Anders Kristensen
Opublikowane w:
Science Advances, Numer 3/5, 2017, Strona(/y) e1602487, ISSN 2375-2548
Wydawca:
Science Advances
DOI:
10.1126/sciadv.1602487
Autorzy:
Nicolai B. Larsen, Sascha E. Liberti, Ivan Vogel, Signe W. Jørgensen, Ian D. Hickson, Hocine W. Mankouri
Opublikowane w:
Proceedings of the National Academy of Sciences, Numer 114/36, 2017, Strona(/y) 9665-9670, ISSN 0027-8424
Wydawca:
National Academy of Sciences
DOI:
10.1073/pnas.1706640114
Autorzy:
Ganesha P. Pitchai, Manuel Kaulich, Anna H. Bizard, Pablo Mesa, Qi Yao, Kata Sarlos, Werner W. Streicher, Erich A. Nigg, Guillermo Montoya, Ian D. Hickson
Opublikowane w:
Nucleic Acids Research, Numer 45/19, 2017, Strona(/y) 11413-11424, ISSN 0305-1048
Wydawca:
Oxford University Press
DOI:
10.1093/nar/gkx792
Autorzy:
Rahul Bhowmick, Ian D Hickson
Opublikowane w:
F1000Research, Numer 6, 2017, Strona(/y) 666, ISSN 2046-1402
Wydawca:
F1000 Research Ltd.
DOI:
10.12688/f1000research.11024.1
Autorzy:
Liqun Ren, Long Chen, Wei Wu, Lorenza Garribba, Huanna Tian, Zihui Liu, Ivan Vogel, Chunhui Li, Ian D. Hickson, Ying Liu
Opublikowane w:
Oncotarget, 2017, ISSN 1949-2553
Wydawca:
Impact Journals
DOI:
10.18632/oncotarget.16940
Autorzy:
Özgün Özer, Rahul Bhowmick, Ying Liu, Ian D. Hickson
Opublikowane w:
Oncotarget, Numer 9/22, 2018, ISSN 1949-2553
Wydawca:
Impact Journals
DOI:
10.18632/oncotarget.24745
Autorzy:
Marcus S. Carstensen, Xiaolong Zhu, Oseze Esther Iyore, N. Asger Mortensen, Uriel Levy, Anders Kristensen
Opublikowane w:
ACS Photonics, Numer 5/5, 2018, Strona(/y) 1665-1670, ISSN 2330-4022
Wydawca:
American Chemical Society
DOI:
10.1021/acsphotonics.7b01358
Autorzy:
Rodolphe Marie, Jonas N. Pedersen, Kalim U. Mir, Brian Bilenberg, Anders Kristensen
Opublikowane w:
Nanoscale, Numer 10/3, 2018, Strona(/y) 1376-1382, ISSN 2040-3364
Wydawca:
Royal Society of Chemistry
DOI:
10.1039/c7nr06016e
Autorzy:
Victoria A. Bjerregaard, Lorenza Garribba, Cynthia T. McMurray, Ian D. Hickson, Ying Liu
Opublikowane w:
Proceedings of the National Academy of Sciences, Numer 115/51, 2018, Strona(/y) 13003-13008, ISSN 0027-8424
Wydawca:
National Academy of Sciences
DOI:
10.1073/pnas.1808377115
Autorzy:
Kata Sarlós, Andreas S. Biebricher, Anna H. Bizard, Julia A. M. Bakx, Anna G. Ferreté-Bonastre, Mauro Modesti, Manikandan Paramasivam, Qi Yao, Erwin J. G. Peterman, Gijs J. L. Wuite, Ian D. Hickson
Opublikowane w:
Nature Structural & Molecular Biology, Numer 25/9, 2018, Strona(/y) 868-876, ISSN 1545-9993
Wydawca:
Nature Publishing Group
DOI:
10.1038/s41594-018-0123-8
Prawa własności intelektualnej
Numer wniosku/publikacji:
16
732524
Data:
2016-06-10
Numer wniosku/publikacji:
20
18050753
Data:
2018-11-12
Wnioskodawca/wnioskodawcy:
LUMICKS BV
Numer wniosku/publikacji:
17
822699
Data:
2017-12-22
Numer wniosku/publikacji:
20
19050071
Data:
2019-02-05
Wnioskodawca/wnioskodawcy:
LUMICKS BV
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