Abstract
Autophagy of the endoplasmic reticulum, or ER-phagy, maintains the homeostasis of the secretory pathway. This is particularly prominent in specialized secretory cells such as the acinar cells of the exocrine pancreas. The role for such a homeostatic pathway during ageing of mammals is modelled best by in vivo genetic or pharmacologic intervention in mice. This is due to the paucity of cellular models that can maintain acinar identity outside of an animal. Here we present methods for isolation of soluble and insoluble protein fractions of ER luminal proteins from the pancreas, alongside RNA. Analysis of these macromolecules allows inference of changes in ER luminal proteostasis upon autophagy-targeted interventions. These methods will likely be more widely applicable, beyond autophagy research.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Levine B, Kroemer G (2019) Biological functions of autophagy genes: a disease perspective. Cell 176:11–42. https://doi.org/10.1016/j.cell.2018.09.048
Klionsky DJ, Abdel-Aziz AK, Abdelfatah S et al (2021) Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 17:1–382. https://doi.org/10.1080/15548627.2020.1797280
Wilkinson S (2020) Emerging principles of selective ER autophagy. J Mol Biol 432:185–205. https://doi.org/10.1016/j.jmb.2019.05.012
Molinari M (2021) ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev Cell 44:217–232. https://doi.org/10.1016/j.devcel.2021.03.005
Smith MD, Wilkinson S (2018) CCPG1, a cargo receptor required for reticulophagy and endoplasmic reticulum proteostasis. Autophagy 14:1090–1091. https://doi.org/10.1080/15548627.2018.1441473
Smith MD, Harley ME, Kemp AJ et al (2018) CCPG1 is a non-canonical autophagy cargo receptor essential for ER-Phagy and pancreatic ER proteostasis. Dev Cell 44:217–232. https://doi.org/10.1016/j.devcel.2017.11.024
Antonucci L, Fagman JB, Kim JY et al (2015) Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress. Proc Natl Acad Sci U S A 112:E6166–E6174. https://doi.org/10.1073/pnas.1519384112
Boj SF, Hwang C, Baker LA et al (2015) Organoid models of human and mouse ductal pancreatic cancer. Cell 160:324–338. https://doi.org/10.1016/j.cell.2014.12.021
Fleming-Martinez AK, Storz P (2019) Mimicking and manipulating pancreatic acinar-to-ductal metaplasia in 3-dimensional cell culture. J Vis Exp. https://doi.org/10.3791/59096
Rovira M, Delsapre F, Massumi M et al (2008) Murine embryonic stem cell-derived pancreatic acinar cells recapitulate features of early pancreatic differentiation. Gastroenterology 135:1301–1310. https://doi.org/10.1053/j.gastro.2008.06.049
Huang L, Desai R, Conrad DN et al (2021) Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids. Cell Stem Cell 28:1091. https://doi.org/10.1016/j.stem.2021.03.022
Acknowledgments
Thanks to Alain Kemp for initial assistance with establishment of the method and to Jennifer Morton for discussions on optimal routes for RNA preservation. Work in the Wilkinson lab is supported by a Cancer Research UK Senior Fellowship (A29576).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Smith, M., Salomo-Coll, C., Muir, M., Wilkinson, S. (2022). Analysis of Pancreatic Acinar Protein Solubility in Autophagy-Deficient Mice. In: Norberg, H., Norberg, E. (eds) Autophagy and Cancer. Methods in Molecular Biology, vol 2445. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2071-7_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2071-7_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2070-0
Online ISBN: 978-1-0716-2071-7
eBook Packages: Springer Protocols