Epithelial cells—the sheets that line blood vessels, the digestive tract, and most other tissues—lose their internal organisation when they become cancerous, and this loss of polarity drives metastasis. Over 80% of cancers arise from epithelia, yet the molecular logic that tells a cell which side is “up” remains poorly understood. This project uses a super-resolution microscopy technique called DNA-PAINT to watch polarity proteins in action inside living tissues, counting individual molecules with nanoscale precision. The researchers have already built the tools to image 8 micrometres deep into a model epithelium, and they have genetically tagged every key polarity protein to track where and when these proteins cluster, interact, and reorganise the actin skeleton that shapes the cell. If successful, this work will reveal the fundamental rules by which epithelial cells maintain their barriers and shapes—knowledge that underpins how tissues develop, repair, and, when disrupted, become cancerous. This is fundamental science: it will not produce a drug or device tomorrow, but understanding polarity at the molecular scale could eventually inform new strategies to detect or block metastasis, much as earlier work on cell adhesion transformed cancer biology.
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Epithelial cells stick together to form the sheets and tubes that make up most tissues, where they act as barriers between compartments (e.g. blood vessels) or between the inside and outside of the body (digestive system). Epithelial sheet formation depends on the polarisation of the cells with their apical surfaces facing outside. This polarity localises the impermeable cell junctions that act as barriers to pathogens and fluids; directs the exocytosis of signals and receptors to the correct side of the epithelium; and controls changes to the sizes of apical, basal and lateral domains as epithelia undergo morphogenesis to form more complex structures. Furthermore, >80% of cancers arise from epithelia and alterations in polarity drive metastasis. Thus, understanding epithelial polarity underlies many aspects of cell physiology, development and tumour biology. Work mainly in Drosophila has identified a conserved set of polarity proteins that define the apical, junctional and lateral domains of epithelial cells and has shown that antagonism between apical and lateral factors determines the relative sizes of each domain. However, there are major gaps in our understanding of how polarity proteins function and the logic of the system is still obscure. This proposal aims to answer these outstanding questions using a super-resolution microscopy technique, called DNA-PAINT. Almost all super-resolution imaging is performed on thin samples using high intensity illumination, and imaging thick samples poses several challenges. We have spent the last 5 years building the tools to overcome these challenges and can now image with 10-20nm resolution at the apical side of our model epithelium, the Drosophila follicular epithelium (~8µm above the coverslip). Furthermore, we have developed techniques and algorithms to count the absolute number of molecules in a structure of interest (qPAINT). We now propose to use DNA-PAINT to address the following questions: 1) Biochemical studies have identified numerous protein-protein interactions between polarity factors, but where and if these interactions occur in vivo is unclear. We have endogenously-tagged every polarity protein with SNAP and HALO so that we can label them with oligonucleotides for DNA-PAINT. Imaging all pairs of polarity proteins will show which proteins interact and reveal where the antagonistic interactions occur. This will also distinguish between competing models for the nature of the boundary between the apical and lateral domains. 2) Our preliminary data and recent work in C. elegans indicate that several polarity proteins cluster at the membrane. We will use qPAINT to determine the stoichiometry of polarity protein clusters and investigate the mechanisms that drive cluster formation. Work on Par-3 suggests that they may form by phase separation. We will use a novel approach to test this hypothesis, in which we vary the amount of a protein and measure its cytoplasmic concentration and cluster size. 3) The polarity system regulates cell shape, which depends on the underlying actin cytoskeleton, but little is known about how polarity factors control actin organisation. We have developed techniques to visualise F-actin at 20nm resolution using the LifeAct peptide. By combining this with DNA-PAINT on polarity proteins and actin regulators, we will determine the relationship between polarity factors and cortical actin organisation. This project will advance our understanding of epithelial polarity, while demonstrating the power of qPAINT to provide quantitative data at the nanoscale. It therefore falls within two BBSRC priority areas: "Understanding the rules of life" and Transformative technologies.
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