Synthetic In Vivo Reconstitution of Asymmetric Cell Division

Change log

Asymmetric cell division (ACD) is the process whereby one dividing cell gives rise to two daughter cells of differing fates. ACD is known to be controlled, at least in part, by protein complexes that reside asymmetrically at the cortex of dividing cells. These asymmetric domains are involved in orienting the division plane, localising proteins important for cell fate, as well as for generating polarity in cytosolic structures important for the dispatch of cell fate determinants. One protein complex of particular interest is the Par complex, which is known to asymmetrically localise to the plasma membrane in many systems. It is known that an asymmetric Par complex is required (in many systems), for orienting the division plane, and for generating asymmetry in the density of microtubules in the anaphase-B central spindle (at least in Drosophila). However, whether the Par complex is sufficient to generate these features, or whether they rely on other polarity pathways, is unknown. In this work, I sought to address this question, by developing two techniques for reimparting cortical polarity back into unpolarised cells (cells that have lost polarity). The first of these techniques relies on protein micropatterning, which is the process by which proteins can be ‘printed’ onto a surface. For successful establishment of this assay, a new system of micropatterning, based on ‘anchoring’ proteins to Fibrinogen (a large glycoprotein complex), was developed. Fibrinogen-anchoring remarkably improves the micropatterning of previously difficult-to-pattern proteins, and gives a universal patterning platform upon which complex, multi-protein patterning experiments can be performed. For the first time, protein micropatterns were used to control the subcellular localisation of cell receptors. Further, with this technology, intracellular plasma membrane asymmetries, akin to those that occur in multicellular organisms, could be reconstituted. The second technology for reconstituting plasma membrane asymmetry was based on novel de novo designed proteins, which, upon mixing, spontaneously assemble into large, two-dimensional lattices. Using quantitative fluorescence microscopy and Atomic Force Microscopy, we demonstrated that these lattices assemble in a near-crystalline fashion on cells. With these proteins, robust asymmetric ‘caps’ of plasma membrane complexes could be generated. Using this technique, I showed that an asymmetric Par complex is indeed sufficient to orient division, and to generate asymmetry in the central spindle. Finally, I showed that these two pathways, while both downstream of an asymmetric Par complex, can be untangled. This represents the most complete reconstitution of asymmetric cell division in symmetrically dividing, unpolarised cells to date. This is the first demonstration that an asymmetric Par complex is sufficient to reinstate polarised signalling and important aspects of ACD in unpolarised cells, and is also the first example of central spindle asymmetry in mammalian cells, revealing this to be a conserved feature of ACD. Further, the technology developed in this study will be of broad use to the field, and will no doubt be used to shed light onto more of the mechanisms controlling cortical symmetry breaking and polarised signalling.

Emmanuel, Derivery
Mitosis, Asymmetric Cell Division, Reconstitution, Protein Design, Micropatterning
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
MRC Studentship Neuberger Studentship from the Max Perutz Foundation
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