Table 1.

The CMA toolkit: overview of the approaches that can be used to measure CMA activity

assay	Principle and description	Key features
CMA reportera	Uses photoconvertible and photoactivable fluorescent reporters that upon conversion change colour or upon activation start to fluoresce. CMA activation is detected as a change in the fluorescence pattern, from cytosolic diffuse to lysosomal punctate pattern, and is quantified as number of puncta per cell	• Pulse-chase experiments • CMA activity measured in intact cells in culture • Test sample size is not a limitation
Lysosomal uptakea	Intact isolated lysosomes pretreated or not with protease inhibitors are incubated with a known CMA substrate, and centrifugation after incubation and immunoblot is used to measure the amount of substrate associated with lysosomes	• In vitro assay • Direct measure of CMA activity • Binding and uptake can be separately measured
Lysosomal protein degradationa	Intact isolated lysosomes are incubated with radiolabelled protein substrates	• In vitro assay • Direct measure of CMA activity • Recapitulates binding, uptake and degradation
Dynamics of CMA translocation complex	Isolated lysosomes are subjected to blue-native electrophoresis (immunoblot) to assess the amount of lysosomal LAMP2A present in the 700 kDa multimeric translocation complex	• In vitro assay • Provides information on CMA dynamics • Useful to determine possible failures in this CMA step
Protein degradation by CMA	Metabolic labelling of intact cells and measurement of the conversion of labelled protein to free-labelled amino acids in the presence or absence of lysosomal inhibitors (to block all forms of autophagy) and of macroautophagy inhibitors	• CMA activity measured in intact cells • Possible compensatory changes • Assesses other forms of autophagy in the same experiment
Immunofluorescence	CMA-active lysosomes are labelled by costaining with antibodies against LAMP2A and HSC70.	• Indirect measurement of CMA • Measures number of lysosomes performing CMA • Useful to determine changes in CMA-related compartments

For comprehensive details on assays and methodology to measure CMA, see REF.¹¹⁷. CMA, chaperone-mediated autophagy; HSC70, heat shock cognate 71 kDa protein; LAMP2A, lysosome-associated membrane protein type 2A.

^aGold-standard assays in the field.