Periodic Reporting for period 3 - CHROMAVISION (Super-resolution visualisation and manipulation of metaphase chromosomes)
Período documentado: 2017-12-01 hasta 2019-05-31
CHROMAVISION aims to develop a pioneering chromosome imaging and manipulation platform to fuel future structural chromosome research. Chromosomal abnormalities are characteristic of many disorders such as cancer, impaired fertility, and neurological disorders. This platform will allow molecular biologists to isolate individual chromosomes from small tissue or cells and deliver them to a super-resolution microscope. Single chromosomes can be brought into focus of the Super-Resolution Correlative Tweezers Fluorescence Microscope (CTFM-SR3D) that is developed in CHROMAVISION. This instrument will for the first time enable 3D, super-resolution, real-time metaphase chromosome observation and manipulation studies under near-physiological conditions. Better imaging and understanding of the chromosomal mechanisms will contribute to our knowledge of the aetiology of human diseases and aid drug discovery. The overall objectives of the proposal are:
1.To develop an integrated and intuitive CTFM-SR3D for chromosome imaging and manipulation that integrates lab-on-a-chip (LOC) microfluidics, multiple-trap optical tweezers and super-resolution fluorescence microscopy
2.To develop and validate production ready prototypes of chromosome labs-on-a-chip for extraction, visualization and actuation of metaphase chromosomes from single cells
3.To develop methods for the quantitative study, in real time, of the 3D structure and dynamics of whole mitotic chromosomes extracted from healthy and diseased single cells
4.To apply the chromosome imaging and manipulation platform to address key questions in biomedical and fundamental chromosome research
Conclusion: Objectives 1, 3 & 4 have been achieved nearly as planned, while objective 2 had to be somewhat modified because chromosome extraction in a lab-on-a-chip setting proved to be very difficult. However, by applying our mitigation plan of extracting chromosomes using more standard bench-top methods and focusing our attention to the development of all-plastic flow chambers, we both managed to avoid any major delays/deviations from the original plans and in fact came, from a commercial point of view, to a better workflow of studying whole metaphase chromosomes. Our general conclusion is that the project was highly successful and has opened new horizons for chromosome research for the coming decade.
1.To develop an integrated and intuitive CTFM-SR3D for chromosome imaging and manipulation that integrates lab-on-a-chip (LOC) microfluidics, multiple-trap optical tweezers and super-resolution fluorescence microscopy
2.To develop and validate production ready prototypes of chromosome labs-on-a-chip for extraction, visualization and actuation of metaphase chromosomes from single cells
3.To develop methods for the quantitative study, in real time, of the 3D structure and dynamics of whole mitotic chromosomes extracted from healthy and diseased single cells
4.To apply the chromosome imaging and manipulation platform to address key questions in biomedical and fundamental chromosome research
Conclusion: Objectives 1, 3 & 4 have been achieved nearly as planned, while objective 2 had to be somewhat modified because chromosome extraction in a lab-on-a-chip setting proved to be very difficult. However, by applying our mitigation plan of extracting chromosomes using more standard bench-top methods and focusing our attention to the development of all-plastic flow chambers, we both managed to avoid any major delays/deviations from the original plans and in fact came, from a commercial point of view, to a better workflow of studying whole metaphase chromosomes. Our general conclusion is that the project was highly successful and has opened new horizons for chromosome research for the coming decade.
WP1
Task 1.1: This task is finished.
Task 1.2: This task is finished.
Task 1.3: This task is finished.
Task 1.4:. This task is finished.
Task 1.5: This task is finished.
Task 1.6: This task is finished.
WP2
Task 2.1:. This task is finished.
Task 2.2: This task is finished.
Task 2.3: During period 3, we worked on using on-chip sonication to include vortexing in the microfluidic chip and disrupt the metaphase cells. We investigated a new device design to maximize the shearing of cells. Despite those effeorts, we did not succeed in an on-chip chromosome extraction. As a mitigation strategy, we resorted to a more classical bench-top chromosome extraction process which turned out to work very well and is easily transferable to other labs.
Task 2.4: DTU made the preliminary steps to integrate a ‘mine field’ to collect metaphase chromosomes extracted from a cell on-chip. Our strategy is to create an array of optical traps by integrating plasmonic-based lenses in the microfluidic chip. DTU has developed a method for laser printing of flat optical components in prefabricated metasurfaces which can be manufactured by production-grade methods and laser reshaping to provide control over optical metasurface functionality. Given the challenges described in task 2.3 we also aimed our efforts in this task to make sure that production of all polymer chips is transferable to LUMICKS, in order to have a complete workflow that can be commercially used.
Task 2.5: We generate active Separase, however, despite multiple test runs with the LOC it proved impossible to separate the sister-chromatids after the metaphase chromosomes were incubated with Separase. We are currently testing whether the reason Separase is unable to break sister chromatid cohesion is because the sisters are interlinked with DNA. We will test that by additionally incubating the chromosomes with DNA unlinking enzymes (Topo IIa, BLM, Topo IIIa, PICH etc., all of which we have purified). This result might be a reflection of the limited understanding in the scientific community of what is really needed to induce in vitro chromosome segregation.
WP3
Task 3.1: This task is finished.
Task 3.2: This task is finished.
Task 3.3: This task is finished.
Task 3.4: We have not been able to follow the segregation of metaphase chromosomes triggered by the introduction of Separase and Topoisomerase II, because it turns out that Separase and Topoisomerase alone are not able to induce chromosome segregation (see also task 2.5).
Task 3.5: This task was dependent on the results of task 3.4 and as such we had to refocus our efforts on other UFB-like structures that validated our instrumental ability to image and manipulated UFBs. The UFB-like structure we studied were nucleoprotein tethers that we could pull out from the telomeres. Such tethers have not been reported before and represent a deeply interesting path for further research because these tethers unravel from the ends of chromosomes in a stick-slip-like fashion and might contain crucial information about the organisation of chromosomes.
WP4
Task 4.1: They were established by all partners and the coordinator.
Task 4.2: All these issues were discussed and agreed upon.
Task 4.3: The procedures are deployed.
Task 4.4: An effective coordinator has established her role well. Communication between partners is effective.
Task 4.5: Many network meetings were organized. A method of collaboration is established.
Task 4.6: A website is launched and actively updated, a flyer is developed, newsletters have been published, an animation is made and we hosted quite a number of public events.
Task 4.7: An exploitation plan is developed and executed.
Task 1.1: This task is finished.
Task 1.2: This task is finished.
Task 1.3: This task is finished.
Task 1.4:. This task is finished.
Task 1.5: This task is finished.
Task 1.6: This task is finished.
WP2
Task 2.1:. This task is finished.
Task 2.2: This task is finished.
Task 2.3: During period 3, we worked on using on-chip sonication to include vortexing in the microfluidic chip and disrupt the metaphase cells. We investigated a new device design to maximize the shearing of cells. Despite those effeorts, we did not succeed in an on-chip chromosome extraction. As a mitigation strategy, we resorted to a more classical bench-top chromosome extraction process which turned out to work very well and is easily transferable to other labs.
Task 2.4: DTU made the preliminary steps to integrate a ‘mine field’ to collect metaphase chromosomes extracted from a cell on-chip. Our strategy is to create an array of optical traps by integrating plasmonic-based lenses in the microfluidic chip. DTU has developed a method for laser printing of flat optical components in prefabricated metasurfaces which can be manufactured by production-grade methods and laser reshaping to provide control over optical metasurface functionality. Given the challenges described in task 2.3 we also aimed our efforts in this task to make sure that production of all polymer chips is transferable to LUMICKS, in order to have a complete workflow that can be commercially used.
Task 2.5: We generate active Separase, however, despite multiple test runs with the LOC it proved impossible to separate the sister-chromatids after the metaphase chromosomes were incubated with Separase. We are currently testing whether the reason Separase is unable to break sister chromatid cohesion is because the sisters are interlinked with DNA. We will test that by additionally incubating the chromosomes with DNA unlinking enzymes (Topo IIa, BLM, Topo IIIa, PICH etc., all of which we have purified). This result might be a reflection of the limited understanding in the scientific community of what is really needed to induce in vitro chromosome segregation.
WP3
Task 3.1: This task is finished.
Task 3.2: This task is finished.
Task 3.3: This task is finished.
Task 3.4: We have not been able to follow the segregation of metaphase chromosomes triggered by the introduction of Separase and Topoisomerase II, because it turns out that Separase and Topoisomerase alone are not able to induce chromosome segregation (see also task 2.5).
Task 3.5: This task was dependent on the results of task 3.4 and as such we had to refocus our efforts on other UFB-like structures that validated our instrumental ability to image and manipulated UFBs. The UFB-like structure we studied were nucleoprotein tethers that we could pull out from the telomeres. Such tethers have not been reported before and represent a deeply interesting path for further research because these tethers unravel from the ends of chromosomes in a stick-slip-like fashion and might contain crucial information about the organisation of chromosomes.
WP4
Task 4.1: They were established by all partners and the coordinator.
Task 4.2: All these issues were discussed and agreed upon.
Task 4.3: The procedures are deployed.
Task 4.4: An effective coordinator has established her role well. Communication between partners is effective.
Task 4.5: Many network meetings were organized. A method of collaboration is established.
Task 4.6: A website is launched and actively updated, a flyer is developed, newsletters have been published, an animation is made and we hosted quite a number of public events.
Task 4.7: An exploitation plan is developed and executed.
We have used any possible opportunity to advertise the mission and activities at CHROMAVISION. These include social media presence, newsletters, an active website, public outreach projects, animations of the project and we just concluded a two-day Symposium and hands-on workshop at the Crick Institute last month (May 2019) with invited speakers and scientists from all over Europe to present CHROMAVISION and metaphase chromosome research in general. In addition, a significant number of publications and patents have been produced. Our big success to be able to routinely manipulate single metaphase chromosomes can be considered beyond the state of the art. When we communicate this achievement within our scientific community, we receive great interest, as was apparent during the concluding two-day Symposium. Furthermore, the success of CHROMAVISION is further highlighted by the fact that LUMICKS was nominated in the category of ‘Best Young SME 2018’ for the H2020 Innovation Radar Prize 2018 and our partner DTU has won the Innovation Radar EU prize 2018 in the ‘Excellent Science’ category for the FET-OPEN project: CHROMAVISION. Many of the technologies developed during the project are now available to Lumicks customers worldwide and thereby have impact not only in the field of Chromosome studies but in the larger and growing field of Dynamic Single Molecule (DSM) analysis.