title: Development of a Kinetic Assay for Late Endosome Movement.
abstract: 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.
authors.affiliation: Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany.
authors.forename: Milan
authors.initials: M
authors.lastname: Esner
authors.affiliation: University of Fribourg, Department of Medicine-Anatomy, Fribourg, Switzerland Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Meyenhofer
authors.affiliation: Biotechnology Center, TU Dresden, Dresden, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Kuhn
authors.affiliation: St Vincent's Centre for Applied Medical Research, Sydney, Australia Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany.
authors.forename: Melissa
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany.
authors.forename: Yannis
authors.initials: Y
authors.lastname: Kalaidzidis
authors.affiliation: Max-Planck Institute of Cell Biology and Genetics, Dresden, Germany bickle@mpi-cbg.de.
authors.forename: Marc
authors.initials: M
authors.lastname: Bickle
mesh_terms: D001331:Automation; D045744:Cell Line, Tumor; D054443:Cell Migration Assays / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D009068:Movement; D015999:Multivariate Analysis; D015739:Nocodazole / Q000737:chemistry; D010641:Phenotype; D025341:Principal Component Analysis; D034622:RNA Interference; D015203:Reproducibility of Results
chemical_list: D049452:Green Fluorescent Proteins; D015739:Nocodazole
keywords: Lamp1; cardiac glycoside; high-content imaging; live cell; tracking