Periodic Reporting for period 3 - CHROMAVISION (Super-resolution visualisation and manipulation of metaphase chromosomes)
Reporting period: 2017-12-01 to 2019-05-31
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.
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.
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.
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.
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.