Quantitative microscopy workflows for the study of cellular receptor trafficking

[摘要] The trafficking and signalling of cellular receptors are complex, intertwined processes with many feedback mechanisms. Confocal microscopy is a powerful tool to study the trafficking of receptors. The aim of this thesis was to report and develop workflows to quantify the spatio-temporal dynamics of receptor trafficking and colocalization using confocal microscopy. Importantly, the workflows should be reproducible and unbiased, as well as automated and accurate. A 4D level set approach is developed to enable accurate cellular segmentation. Temporal constraints are introduced to further improve segmentation accuracy. This novel approach is thoroughly validated, and statistically significant performance increase over equivalent 2D and 3D approaches is demonstrated. We present a confocal microscopy based RNAi depletion screen. Specifically, quantitative workflows to identify genes which perturb the trafficking of receptor are described. Finally, a critical review of current approaches to the quantification of colocalization between receptors and endosomes is presented. Improvements to existing techniques and complete workflows are provided for 4D data (3D time-lapse).Together the described protocols provide a complete microscopy based platform to identify and investigate regulators of receptor signalling and trafficking.

[发布机构] University:University of Birmingham;Department:School of Chemistry

[关键词] Q Science;QD Chemistry