Quantifying receptor trafficking and colocalization with confocal microscopy

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Quantifying receptor trafficking and colocalization with confocal microscopy. / Pike, Jeremy; Styles, Iain B; Rappoport, Joshua Z; Heath, John K.

In: Methods, Vol. 115, 15.03.2017, p. 42–54.

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@article{58c65c6695a249efba19068ae244748b,
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.",
keywords = "Colocalization , Trafficking , Endocytosis , Confocal , Receptor , EGFR",
author = "Jeremy Pike and Styles, {Iain B} and Rappoport, {Joshua Z} and Heath, {John K}",
note = "Copyright {\textcopyright} 2017. Published by Elsevier Inc.",
year = "2017",
month = mar,
day = "15",
doi = "10.1016/j.ymeth.2017.01.005",
language = "English",
volume = "115",
pages = "42–54",
journal = "Methods",
issn = "1046-2023",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Quantifying receptor trafficking and colocalization with confocal microscopy

AU - Pike, Jeremy

AU - Styles, Iain B

AU - Rappoport, Joshua Z

AU - Heath, John K

N1 - Copyright © 2017. Published by Elsevier Inc.

PY - 2017/3/15

Y1 - 2017/3/15

N2 - 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.

AB - 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.

KW - Colocalization

KW - Trafficking

KW - Endocytosis

KW - Confocal

KW - Receptor

KW - EGFR

U2 - 10.1016/j.ymeth.2017.01.005

DO - 10.1016/j.ymeth.2017.01.005

M3 - Article

C2 - 28131869

VL - 115

SP - 42

EP - 54

JO - Methods

JF - Methods

SN - 1046-2023

ER -