Developing qPAINT to count molecules in polarity complexes and measure secretory cargo flux in epithelial cells.

The aim of this project is to develop and validate a new microscopy technique called qPAINT that counts the number of protein molecules in specific structures or complexes inside a cell. This is important, because it can reveal where proteins work together to perform biological functions and will provide quantitative data on how many copies of each protein a structure contains, which is essential for understanding how these proteins interact and for building models of biological processes. To test qPAINT, we will count the number of copies of specific proteins in the nuclear pores, which are the channels that regulate the movement of molecules in and out of the cell nucleus. We have chosen the nuclear pore because it is one of the few structures in the cell where the number of copies of each protein is already known. This will allow us to verify that qPAINT produces accurate data and to calibrate our system so that we can count the number of molecules in other structures whose composition is unknown.

Once validated, we will use qPAINT to analyse the spatial organisation of epithelial cells, the most common cell-type in our bodies. Epithelial cells stick together to form the sheets or tubes that make up most of our organs, where they act as barriers between compartments (e.g. blood vessels; secretory glands) or between the inside and outside of the body (skin, digestive system and lungs). The formation of epithelial sheets depends on the coordinated polarisation of the cells, so that all have the same surface (apical) facing the outside and the inside (basal). Epithelial cells also control the movement of molecules from one side of the sheet to the other. For example, intestinal cells absorb food from the gut, while the cells of the mammary gland secrete milk proteins apically into mammary ducts. Disruption of apical-basal polarity can lead to several types of disease. For example, more than 80% of cancers arise from epithelial tissues and one of their hallmarks is a progressive loss of polarity, which correlates with tumour malignancy.

The polarity proteins that make the apical and basal sides of epithelial cells different are largely known, but we have limited information about where these proteins interact with each other and how much of each protein is present. Recent research also suggests that some of these polarity proteins form clusters, but the mechanisms that drive cluster formation are unclear. We will investigate how these clusters form using qPAINT to count the number of molecules of each polarity protein in clusters in different regions of the cell, as well as their overall levels in the cytoplasm.

Another key feature of epithelial cells is their ability to target the secretion of proteins to different sides of the cell. Secreted proteins are made in the endoplasmic reticulum and then move to the Golgi complex, where they are packaged into small vesicles surrounded by lipid membranes that are transported to the cell surface. We have developed a system in which we can release a pulse of a labelled secreted protein from the endoplasmic reticulum and watch its movement to the cell surface. We now plan to use qPAINT to count the number of molecules in the Golgi, vesicles and at the cell surface at different times after release. This will allow us to calculate how many cargo molecules are packaged into a vesicle and how quickly each secreted cargo moves to the cell surface. By releasing two cargoes that traffic to different places simultaneously, we will also measure how and where secreted proteins are sorted into the vesicles that deliver them to the correct region of the cell surface. In conclusion, this project will advance our quantitative understanding of how cells are polarised and how the secretory system sorts and transports proteins to the cell surface, while demonstrating how qPAINT can be used to count the number of molecules in any structure or region of interest in a cell.

This project aims to validate qPAINT as a method for counting the number of protein molecules in a defined structure or complex. We have generated Drosophila lines in which endogenous proteins of interest are fused to SNAP or HALO tags. After conjugating a short docking strand oligonucleotide to the tag, the protein can then be imaged using the complementary imager oligonucleotide fused to a fluorescent dye (DNA-PAINT). The predictable kinetics of the DNA hybridisation that creates blinks in DNA-PAINT means that the number of molecules in a region is proportional to the frequency of blinks (qPAINT).

We will use 3 nucleoporins (Nups) to test qPAINT in the follicle cells, as the number of Nup molecules in the nuclear pore is known. Accurate counting requires knowing the labeling efficiency and the mean dark time between blinks for a single molecule (td1). We will measure the former using western blots, since the protein migrates more slowly when conjugated to the docking strand and determine td1 in cells in which some nuclear pores contain only one labelled Nup. We will then use qPAINT to count the number of each Nup in the pore and compare this to the ground truth from CryoEM.

Once validated, we will use two colour qPAINT to determine the composition and stoichiometry of epithelial polarity protein complexes in different regions of the follicle cells. To test if these clusters form by phase separation, we will measure the cytoplasmic concentration of each factor and its partitioning into clusters, while varying the dosage of each cluster component.

We will also use qPAINT to quantify flux through the secretory pathway. After synchronous release of tagged cargoes from the ER, we will fix and count cargo molecules in the Golgi, post-Golgi vesicles and at the plasma membrane at different time points. This will reveal the rate at which apical, lateral and basal cargoes are secreted, the efficiency of cargo sorting and the number of molecules in each vesicle.