Project Reference: TRG27-GRD-DS1

Project title: Dissecting epithelial polarity using acute protein depletion

Project outline

Most animal tissues are composed of epithelial cells that form sheets that act as barriers between compartments. The formation of epithelial monolayers depends on the apical-basal polarisation of each cell, and this polarity underlies all aspects of epithelial biology. We know most factors that generate apical-basal polarity in epithelia, but their functions are not well understood, because null mutations cause a complete loss of polarity, which is uninformative. Furthermore, protein levels only decay slowly after the induction of a mutant clone or RNAi, making it hard to distinguish the direct effects of protein loss from indirect effects or compensatory mechanisms. These limitations can be overcome by acutely inhibiting a polarity factor and studying which effects occur first and are direct responses and which take longer and are secondary. 

The goal of the project is to develop a general method for acute protein knock down in Drosophila, in which the protein of interest is rapidly sequestered at the mitochondrial surface using the Catchfire technique. Once the approach has been proven to work on a test target (aPKC), it will be applied to the lateral polarity factors and tumour suppressors, Discs large (Dlg) and Scribble, whose functions are largely unknown. Making movies of tagged polarity factors after the treatment should reveal which are affected first and how the knock-down alters epithelial cell mechanics and cell shape. We will also perform super-resolution microscopy to examine how the nanoscale organisation of cytoskeleton changes over time and how the stoichiometry and localisation of polarity protein clusters is affected. These experiments should reveal the functions of Scribble and Dlg and distinguish between two competing models for how antagonism between apical and lateral polarity factors occurs. If the Catchfire approach is not effective, other options include using the auxin-dependent degron system or the traditional knocked sideways approach.

Principal Supervisor

Professor Daniel St Johnston

Email address: ds139@cam.ac.uk

Department / Partner Institute: Gurdon Institute

Website: https://www.gurdon.cam.ac.uk/people/daniel-st-johnston/

BBSRC strategic theme

Understanding the rules of life