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Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches

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Membrane Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2473))

Abstract

The endosomal recycling pathway plays a crucial role in diverse physiologically important biological processes such as cell-to-cell signaling, nutrient uptake, immune response, and autophagy. A selective subset of these recycling cargoes, mostly transmembrane proteins, is retrieved from endosomes to the trans-Golgi network (TGN) by a retrograde transport process. Endosome-to-TGN retrograde trafficking is crucial for maintaining cellular homeostasis and signaling by preventing proteins and lipids from degradation in the lysosome. Many of the membrane sorting machinery, such as the retromer complex and sorting nexins (SNXs) are involved in endosomal retrieval and recycling of various transmembrane proteins. Recent technological advances in the resolution of light microscopy and unbiased analytical approaches in quantitative image analysis enable us to explore and understand the regulation of membrane trafficking pathways in greater detail. In this chapter, we describe quantitative imaging-based methods for analyzing the roles of proteins involved in the retrograde trafficking in retromer dependent or independent fashion, using cation-independent mannose-6-phosphate receptor (CIM6PR) as an example.

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Acknowledgments

We sincerely thank Prof. M.N.J Seaman for providing us HeLa cells stably expressing GFP-Golph3 and CD8-CIM6PR reporter construct. We also thank Prof. Yannis Kalaidzidis for his tremendous help in image quantifications with MotionTracking software, and Angelika Giner for providing us with endogenous EEA1 antibody and the protocol for preparing the Mowiol mounting media. We also thank Dr. Priyanka Sharma for her help with the image quantification write-up.

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Authors and Affiliations

  1. Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France

    Amulya Priya

  2. Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal, India

    Sunando Datta

Authors
  1. Amulya Priya
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  2. Sunando Datta
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Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO, USA

    Jingshi Shen

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Priya, A., Datta, S. (2022). Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches. In: Shen, J. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 2473. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2209-4_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2209-4_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2208-7

  • Online ISBN: 978-1-0716-2209-4

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