Abstract
Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, including strategies for signal isolation and statistical testing. By calculating the Manders and Pearson coefficients, both co-occurrence and correlation can be assessed. A statistically significant decrease in the level of ligand induced co-occurrence between EGFR and rab5 positive endosomes is demonstrated for both the AG1478 and Dynasore treated cells relative to a control. Finally, a strategy for the visualisation of co-occurrence is presented, which provides an unbiased alternative to colour overlays.
Highlights
The signalling and trafficking of cellular receptors are highly interlinked processes [1,2,3]
We demonstrate the effectiveness of the described workflows, and show that both drug treatments perturb epidermal growth factor receptor (EGFR) trafficking and colocalization with rab5 positive endosomes
Custom image analysis solutions are typically required for the analysis of subcellular signal distribution and colocalization
Summary
The signalling and trafficking of cellular receptors are highly interlinked processes [1,2,3]. The hypothesis of signalling endosomes, for which there is extensive evidence, implies that the subcellular location of activated receptor triggers distinct signalling responses [3,4,5,6,7,8]. Fluorescence microscopy is commonly used to study ligand induced changes to the quantity of receptor located at the plasma membrane [13], and colocalization with subcellular structures, such as endosomes [14]. Ligand treatment triggers receptor internalisation and a decrease in the SNR for the EGFR-EGFP channel at the plasma membrane. Validation of the segmentation protocol was performed using a reference produced by blind manual segmentation of EGFR-EGFP and transmission images.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
