Visualising Asymmetry: Monitoring the segregation of proteins important in neurogenesis
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2016-06-22
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en
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Asymmetric cell division is an essential mechanism in the process of neurogenesis, which produces cells with different fates. Currently, our knowledge of which cellular components are subject to asymmetric segregation is lacking. Fatty acid synthase (FASN) has an important function in dividing stem cells but not in non-dividing cells, which makes it a good candidate to be unevenly segregated to the stem cell. Thus, I used the iCOUNT system to investigate the segregation pattern of FASN in dividing cells. The iCOUNT is a novel genetic tool that relies on an inducible colour switch that facilitates the investigation of asymmetrical segregation of intracellular components. I was able to tag FASN with the iCOUNT construct using CRISPR/Cas9, allowing me to determine that FASN is localised to the cytoplasm and segregated mostly symmetrically in mouse embryonic stem cells and neural stem cells that were tested. However, some asymmetry was observed with time, likely due to an accumulation of damage. To determine whether this asymmetry correlates with intrinsic cellular asymmetry I aimed to tag centrosomal components with the iCOUNT construct allowing the distinction between young and old centrosomes. Visualising centrosomes by tagging Centrin1 was unsuccessful. However, by targeting Pericentrin (PCNT), an integral component of the pericentriolar material, the centrosomes and their distinct stages were clearly visible. Further investigation is necessary to characterise PCNT inheritance, before it can be used to answer some of the fundamental questions of asymmetry. Finally, I developed an improved iCOUNT system utilizing a double colour switch based on two serial recombination events enabling the visualization of the tagged protein for more cell divisions (called miCOUNT). These data show the potential of the iCOUNT/miCOUNT system as a tool to visualise asymmetric segregation of various proteins in real time.
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Faculteit der Sociale Wetenschappen