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. 2014;10(11):2087-96.
doi: 10.4161/15548627.2014.973338.

Defining and measuring autophagosome flux—concept and reality

Ben Loos  1 , André du Toit, Jan-Hendrik S Hofmeyr
Affiliations

Defining and measuring autophagosome flux—concept and reality

Ben Loos et al. Autophagy. 2014.

Abstract

The autophagic system is involved in both bulk degradation of primarily long-lived cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and a number of methods are currently utilized to assess autophagic flux. However, despite major advances in measuring various molecular aspects of the autophagic machinery, we remain less able to express autophagic flux in a highly sensitive, robust, and well-quantifiable manner. Here, we describe a conceptual framework for defining and measuring autophagosome flux at the single-cell level. The concept discussed here is based on the theoretical framework of metabolic control analysis, which distinguishes between the pathway along which there is a flow of material and the quantitative measure of this flow. By treating the autophagic system as a multistep pathway with each step characterized by a particular rate, we are able to provide a single-cell fluorescence live-cell imaging-based approach that describes the accurate assessment of the complete autophagosome pool size, the autophagosome flux, and the transition time required to turn over the intracellular autophagosome pool. In doing so, this perspective provides clarity on whether the system is at steady state or in a transient state moving towards a new steady state. It is hoped that this theoretical account of quantitatively measuring autophagosome flux may contribute towards a new direction in the field of autophagy, a standardized approach that allows the establishment of systematic flux databases of clinically relevant cell and tissue types that serve as important model systems for human pathologies.

Keywords: CMA, chaperone-mediated autophagy; GFP, green fluorescent protein; J, flux; LC3, microtubule-associated protein 1 light chain 3; TEM, transmission electron microscopy; nA, number of autophagosomes; τ, transition time.

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Figures

Figure 1.
Figure 1.
Defining autophagosome flux. A, autophagosome; AA, amino acids; AL, autolysosome; L, lysosome; P, phagophore; v, rate.
Figure 2.
Figure 2.
From a micrograph to a number. (A) Live-cell imaging of mouse embryonic fibroblasts stably expressing GFP-LC3 reveals accumulation of autophagosomes over time in the presence of bafilomycin A1. (B) Software solutions exist to assist in automated counting of the complete autophagosome pool. Scale bar: 20 μm (A) and 10 μm (B).
Figure 3.
Figure 3.
Generation of progress curves I. (A) Counting of autophagosomes (nA) over time under control conditions is required to show whether the system is at steady state. (B) The quantitative measurement of the basal autophagosome flux, J, at steady state expressed as autophagosomes produced/cell/time. Here the autophagosome pool size differs (nA =  30, left and nA  =  5, right), while the basal flux J  =  5 is equal in both systems.
Figure 4.
Figure 4.
Generation of progress curves II. (A) The autophagosome pool is the same (nA  =  5), while the basal flux J differs for the 2 systems (left J  =  15; right J  =  5). (B) A system with functional autophagosome synthesis (left) defined by its autophagosome pool size nA  =  5 and a non-zero autophagosome flux J  =  15 compared to a completely dysfunctional autophagosome maturation (right) with nA  =  5 but no autophagosome flux (J  =  0).
Figure 5.
Figure 5.
A comparison of 3 cellular systems that differ in terms of all 3 autophagic steady-state variables: flux J, autophagosome pool size nA, and transition time τ.
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