title: Quantifying receptor trafficking and colocalization with confocal microscopy.
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.
authors.affiliation: PSIBS Doctoral Training Centre, School of Chemistry, University of Birmingham, Birmingham B15 2TT, UK; School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK. Electronic address: jeremy.pike@cruk.cam.ac.uk.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Pike
authors.affiliation: School of Computer Science, University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Iain B
authors.initials: IB
authors.lastname: Styles
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK; Center for Advanced Microscopy and the Nikon Imaging Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Rappoport
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: John K
authors.initials: JK
authors.lastname: Heath
mesh_terms: D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D021381:Protein Transport / Q000187:drug effects; D011799:Quinazolines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D020032:Tyrphostins / Q000494:pharmacology; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D049452:Green Fluorescent Proteins; D006835:Hydrazones; D008164:Luminescent Proteins; D011799:Quinazolines; D011993:Recombinant Fusion Proteins; D020032:Tyrphostins; D020696:rab5 GTP-Binding Proteins; D000097573:Red Fluorescent Protein; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; C485184:enhanced green fluorescent protein; C101044:RTKI cpd; C512478:EGFR protein, human; C427755:RAB5C protein, human
keywords: Colocalization; Confocal; EGFR; Endocytosis; Receptor; Trafficking