A single protein acts as a dimmer switch inside cells, and turning it down just a notch makes the cells squeeze themselves into new shapes. The protein, called aPKC, controls which other proteins it modifies based on how active it is—a property that has been conserved across evolution. When aPKC activity drops slightly, it stops modifying a low-priority target called Yurt, and Yurt then moves to the cell’s top surface and triggers a contraction. This is the first known example of a polarity protein directly driving a shape change in a tissue. This matters because scientists do not understand how sheets of epithelial cells—which line most organs—bend and fold to form tubes and other structures during development. The project will test whether aPKC acts as a homeostatic sensor that responds to tissue stretching, and whether its activity is deliberately turned down during normal development to trigger shape changes. This is fundamental curiosity-driven science. It will reveal basic rules of how animal bodies are built. Understanding this pathway could eventually inform research into cancer metastasis, because cells undergoing epithelial-to-mesenchymal transitions—a step in metastasis—show the same hallmarks of mildly reduced aPKC activity.
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Most animal tissues are composed of sheets of epithelial cells that are polarised along their apical-basal axes by conserved polarity factors that define their apical, junctional and lateral domains. Coordinated cell shape changes in epithelial sheets drive morphogenesis to form more complex structures. For example, apical constriction bends epithelial sheets to drive to tube formation. Epithelial morphogenesis therefore requires changes to the relative sizes of the cells' apical, lateral and basal domains, but how polarity factors regulate and are regulated by these changes is unknown. We discovered that the substrates of the key effector of apical domain identity, atypical protein kinase C (aPKC) differ 500-fold in their sensitivity to aPKC inhibition. Thus, like the growth regulator TORC1 and the cell cycle kinase Cdk1, aPKC functions as a rheostat that phosphorylates fewer substrates as its activity decreases, a property that is evolutionary conserved. We found that a small reduction in aPKC activity blocks only the phosphorylation of its lowest affinity substrate Yurt (EPB41L5/Lulu1 in mammals), leading to apical Yurt localisation and apical constriction, the first example of a morphogenetic process driven by a polarity protein. Mild aPKC inhibition also induces endocytosis of the apical determinant Crumbs, which is an early step in epithelial to mesenchymal transitions (EMT). Thus, aPKC modulation provides an attractive mechanism to link the polarity system to morphogenesis. This proposal sets out to determine how aPKC regulates Yurt and Crumbs and the role of this pathway in Drosophila development. 1) We will investigate the mechanism of Yurt-mediated apical constriction, as identifying the components of this novel myosin activation pathway will provide clues about where it functions. 2) We will identify the aPKC substrate that regulates Crumbs endocytosis and map the aPKC phosphorylation sites in it and Yurt. 3) The most rigorous test of aPKC's function as a rheostat is to turn its low affinity substrates into high affinity substrates, so they are still phosphorylated when aPKC is less active. We will therefore change the relevant sites in Yurt into high affinity sites and examine the phenotypes. As an alternative strategy, we will prevent the apical recruitment of Yurt by Crumbs by mutating the Yurt-binding site. We will then use these tools test the following hypotheses: i) This pathway could function as a homeostatic mechanism to maintain apical domain size in response to stretching, as stretching should dilute apical aPKC activity and induce constriction. We will test this in collaboration with Yanlan Mao (UCL) by stretching wing imaginal discs and examining Yurt localisation. ii) aPKC activity could be developmentally down-regulated to promote apical constriction. We will test this in the salivary placode, which is an excellent candidate for such a mechanism because it has lower levels of aPKC along its external periphery, where a contractile myosin cable forms. iii) Cells undergoing EMT apically constrict and endocytose Crumbs, both hallmarks of mildly reduced aPKC activity. We will test in delaminating neuroblasts whether EMT is coordinated by reducing aPKC activity. This project investigates a new, conserved pathway that controls epithelial morphogenesis and links the polarity system to cell shape control. It will therefore inform research in many areas of invertebrate and vertebrate development. It may also be relevant to medical research, since EMT contributes to cancer metastasis. It falls within the BBSRC's strategic objective of "understanding the fundamental rules of life".
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