Advantages and Limitations of Different p62-Based Assays for Estimating Autophagic Activity in Drosophila

Abstract

Levels of the selective autophagy substrate p62 have been established in recent years as a specific readout for basal autophagic activity. Here we compared different experimental approaches for using this assay in Drosophila larvae. Similar to the more commonly used western blots, quantifying p62 dots in immunostained fat body cells of L3 stage larvae detected a strong accumulation of endogenous p62 aggregates in null mutants for Atg genes and S6K. Importantly, genes whose mutation or silencing results in early stage lethality can only be analyzed by microscopy using clonal analysis. The loss of numerous general housekeeping genes show a phenotype in large-scale screens including autophagy, and the p62 assay was potentially suitable for distinguishing bona fide autophagy regulators from silencing of a DNA polymerase subunit or a ribosomal gene that likely has a non-specific effect on autophagy. p62 accumulation upon RNAi silencing of known autophagy regulators was dependent on the duration of the knockdown effect, unlike in the case of starvation-induced autophagy. The endogenous p62 assay was more sensitive than a constitutively overexpressed p62-GFP reporter, which showed self-aggregation and large-scale accumulation even in control cells. We recommend western blots for following the conversion of overexpressed p62-GFP reporters to estimate autophagic activity if sample collection from mutant larvae or adults is possible. In addition, we also showed that overexpressed p62 or Atg8 reporters can strongly influence the phenotypes of each other, potentially giving rise to false or contradicting results. Overexpressed p62 aggregates also incorporated Atg8 reporter molecules that might lead to a wrong conclusion of strongly enhanced autophagy, whereas expression of an Atg8 reporter transgene rescued the inhibitory effect of a dominant-negative Atg4 mutant on basal and starvation-induced autophagy.

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects

“…Filters were probed with anti-Ref(P)2/SQSTM1/p62 (rabbit, 1:2500), 63 anti-EDTP (rat, 1:1000, described in this study), anti-rabbit IgG alkaline phosphatase (1:1000, Sigma, A3687), anti-mouse IgG alkaline phosphatase (1:1000, Sigma, A8438) and anti-rat IgG alkaline phosphatase (1:1000, Sigma, A5153), and developed by NBT-BCIP solution (Sigma, 72091).…”

Coordination of autophagosome–lysosome fusion and transport by a Klp98A–Rab14 complex in Drosophila

“…To ask whether the vesicle fusion defect observed in Klp98A-depleted cells ultimately affects the overall rates of autophagic degradation, we examined the levels of GFP-tagged Ref(2)P (a p62 ortholog), a selective autophagy substrate whose levels are highly sensitive to autophagic flux (Chang and Neufeld, 2009;Lindmo et al, 2008;Nezis et al, 2008;Pircs et al, 2012). In control cells, GFP-Ref(2)P protein levels rapidly decreased during starvationinduced autophagy.…”

Drosophila Atg16 promotes enteroendocrine cell differentiation via regulation of intestinal Slit/Robo signaling

“…S9A). Ref(2)P (also known as p62) is a selective autophagy cargo, and its levels inversely correlate with autophagic degradation in Drosophila (Nagy et al, 2015;Nezis et al, 2008;Pircs et al, 2012 aggregates, whereas its level was comparably low in control and Atg16…”

“…Briefly, 20 guts were isolated and homogenized in Laemmli sample buffer (Sigma) containing aprotinin, leupeptin and PMSF to avoid protein degradation. Primary antibodies were rat anti-Atg16/1 (1:5000) , rabbit anti-p62 (1:5000) (Pircs et al, 2012), rabbit anti-ubiquitin (1:500; DAKO, Z0458) and mouse anti-α-tubulin (1:1000; DSHB, AA4.3-s). Secondary antibodies were alkaline phosphatase-conjugated goat anti-rat, anti-rabbit and anti-mouse (1:5000; Millipore).…”

“…Primary antibodies used were rabbit anti-p62 (1:2000) (Pircs et al, 2012), chicken anti-GFP (1:1500; Invitrogen, A-10262), mouse anti-Delta (1:100; DSHB, C594.9B), mouse anti-Slit (1:100; DSHB, C555.6D), rabbit anti-phosphohistone H3 (1:300; Millipore, 06-570), mouse anti-Armadillo (1:50; DSHB, N2 7A1), mouse anti-Prospero (1:100; DSHB, MR1A), rat anti-mCherry (1:300) (Takáts et al, 2013), rabbit anti-HA (1:50; Sigma-Aldrich, H6908), mouse anti-β-galactosidase (1:100; DSHB, 40-1a), rabbit anti-Allatostatin C and rabbit anti-DH31 [both 1:200; kindly provided by Jan Veenstra (Veenstra et al, 2008)]; and secondary antibodies (all 1:1500; Invitrogen) were Alexa Fluor 488 anti-chicken (A11039), Alexa Fluor 586 anti-rabbit (A11036), Alexa Fluor 568 anti-mouse (A11031), Alexa Fluor 647 antirabbit (A21245) and Alexa Fluor 568 anti-rat (A11077).…”