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Quantitative Co-Localization and Pattern Analysis of Endo-Lysosomal Cargo in Subcellular Image Cytometry and Validation on Synthetic Image Sets

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Lysosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1594))

Abstract

Late endosomes and lysosomes (LE/LYSs) play a central role in trafficking of endocytic cargo, secretion of exosomes, and hydrolysis of ingested proteins and lipids. Failure in such processes can lead to lysosomal storage disorders in which a particular metabolite accumulates within LE/LYSs. Analysis of endocytic trafficking relies heavily on quantitative fluorescence microscopy, but evaluation of the huge image data sets is challenging and demands computer-assisted statistical tools. Here, we describe how to use SpatTrack (www.sdu.dk/bmb/spattrack), an imaging toolbox, which we developed for quantification of the distribution and dynamics of endo-lysosomal cargo from fluorescence images of living cells. First, we explain how to analyze experimental images of endocytic processes in Niemann Pick C2 disease fibroblasts using SpatTrack. We demonstrate how to quantify the location of the sterol-binding protein NPC2 in LE/LYSs relative to cholesterol -rich lysosomal storage organelles (LSOs) stained with filipin. Second, we show how to simulate realistic vesicle patterns in the cell geometry using Markov Chain Monte Carlo and suitable inter-vesicle and cell-vesicle interaction potentials. Finally, we use such synthetic vesicle patterns as “ground truth” for validation of two-channel analysis tools in SpatTrack, revealing their high reliability. An improved version of SpatTrack for microscopy-based quantification of cargo transport through the endo-lysosomal system accompanies this protocol.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark

    Frederik W. Lund & Daniel Wüstner

  2. Department of Biochemistry, Weill Medical College of Cornell University, New York, NY, 10065, USA

    Frederik W. Lund

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  1. Frederik W. Lund
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  2. Daniel Wüstner
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Correspondence to Daniel Wüstner .

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Editors and Affiliations

  1. Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

    Karin Öllinger

  2. Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden

    Hanna Appelqvist

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Lund, F.W., Wüstner, D. (2017). Quantitative Co-Localization and Pattern Analysis of Endo-Lysosomal Cargo in Subcellular Image Cytometry and Validation on Synthetic Image Sets. In: Öllinger, K., Appelqvist, H. (eds) Lysosomes. Methods in Molecular Biology, vol 1594. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6934-0_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6934-0_6

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  • Print ISBN: 978-1-4939-6932-6

  • Online ISBN: 978-1-4939-6934-0

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