Quantitative Imaging of Protein Complexes using TIRF Microscopy

The dynamic composition of protein complexes can have a profound impact on their function. However, conventional pull-down assays cannot provide dynamic information, and sample preparation may affect complex stability. Here, we combine total internal reflection (TIRF) imaging with quantitative image and data analysis to examine the stoichiometry of protein complexes as well as the dynamics of protein associations. Fluorescently labeled proteins are immobilized on glass coverslips, and perfused with other fluorescently tagged protein or small molecules. Complexes are imaged on a TIRF microscope to selectively illuminate bound proteins. We determine copy number and relative positions of fluorescent proteins inside complexes by counting photobleaching vents, using computational super-resolution and mixture-model fitting algorithms for spot detection. We then statistically correct the results for experimental artefacts, such as expression levels and pre-bleaching of fluorophores, which can reach levels of 20%. We further determine binding kinetics by measuring dwell time of fluorescently labeled proteins on immobilized substrates using the same computational approach. Together these methods have allowed us to determine that CENP-A exists in octameric nucleosomes throughout the cell cycle, that myosin adopts multiple conformations on the cortex of the C. elegans zygote that are differentially sensitive to perturbations, and that the interaction of KNL2 with CENP-A in depends on its phosphorylation and its Myb-domain structure.