Measuring forces during DNA segregation

Ali Nick Maleki

Microtubules are involved in DNA segregation in higher eukaryotes. During mitosis bundles of microtubules called k-fibers attach to the chromosomes via kinetochore and pull sister chromatids apart. We present an experimental approach to estimate the forces involved in this process through the extension of a fluorescent spring-shaped DNA origami. Optical readout of the spring extension enabled recording of force production simultaneously with single-molecule fluorescence of proteins getting recruited to the site of force generation. Additionally, this work aims to reconstitute the process of DNA congression and segregation and find minimal number of components required for such function. In this scheme, DNA origami nanospring was used as a cargo with multiple sticking-out handles on which various microtubule associated proteins (MAPs) could assemble. We used kinesin1 motors as means of active transport towards the plus end of microtubule and Ndc80 complex trimers to utilize the forces generated by depolymerizing microtubules to segregate the DNA.

Figure 1: Schematic representation of nanospring with incorporated kinesin motors (red), fluorescent oligonucleotides (blue) and Ndc80 trimers (yellow).
Figure 2: Plus end directed motility and tip tracking of free nanospring-Ndc80-kinesin complex
Figure 3: Measuring forces produced by kinesin motors and depolymerizing forces captured by Ndc80 trimers.

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