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Supply and Demand Analysis of Autophagy

  • André du ToitEmail author
  • Ben Loos
  • Jan Hendrik S. Hofmeyr
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)

Abstract

Autophagy is an intracellular protein degradation pathway that plays a vital role in cellular homeostasis. It maintains cellular function through proteostasis and the removal of unused and harmful proteins and organelles. Moreover, it also serves as an adaptive response to metabolic perturbations. Deviation in autophagy activity has been linked to the progression of several pathologies, including neurodegenerative diseases. Preclinical trials have shown that modulating autophagy holds great promise in treating neurodegenerative diseases by clearing toxic protein aggregates. The success of autophagy modulating therapies requires extensive knowledge of the molecular machinery and, importantly, an in-depth understanding of the underlying systems properties of the autophagy system. A computational approach provides a powerful platform to interrogate and analyze the regulation, control, and behavior of reaction networks. However, the complexity of interactions involved in the autophagy pathway makes it challenging to isolate and characterize individual components. By reducing the autophagy process to a supply-demand system in which autophagosome synthesis (supply) and autophagosome degradation (demand) are linked by the autophagosomes, it is possible to determine the control of the supply and demand over the steady-state autophagosome flux and autophagosome concentration. In this chapter, we describe a methodology to perform supply and demand analysis of the autophagy system, the experimental procedure to measure the autophagy variables, and the use of the supply-demand framework to determine the distribution of flux and concentration control.

Key words

Autophagy Autophagosome flux Supply-demand analysis Elasticity coefficient Control coefficient Autophagosomes Fluorescence microscopy 

Notes

Acknowledgements

We acknowledge financial support from the South African National Research Foundation (NRF), the South African Medical Research Council (SAMRC) as well as the Cancer Association of South Africa (CANSA). All microscopy work was performed at the Central Analytical Facility (CAF), Cell Imaging Unit, Stellenbosch University, South Africa.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • André du Toit
    • 1
    • 2
    Email author
  • Ben Loos
    • 2
  • Jan Hendrik S. Hofmeyr
    • 1
  1. 1.Department of BiochemistryStellenbosch UniversityStellenboschSouth Africa
  2. 2.Department of Physiological SciencesStellenbosch UniversityStellenboschSouth Africa

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