Development of a kinetic assay for late endosome movement

Esner, Milan; Meyenhofer, Felix; Kuhn, Michael; Thomas, Melissa; Kalaidzidis, Yannis; Bickle, Marc

doi:10.1177/1087057114524278

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Journal article

Development of a kinetic assay for late endosome movement

Esner, Milan Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic - Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany
Meyenhofer, Felix University of Fribourg, Department of Medicine–Anatomy, Switzerland - Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany
Kuhn, Michael Biotechnology Center, TU Dresden, Dresden, Germany
Thomas, Melissa St Vincent’s Centre for Applied Medical Research, Sydney, Australia - Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany
Kalaidzidis, Yannis Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany
Bickle, Marc Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany

01.08.2014

Published in:

Journal of Biomolecular Screening. - 2014, vol. 19, no. 7, p. 1070–1078

English Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

Faculty

Faculté des sciences et de médecine

Department

Département de Médecine

Language