The Dynamic Membrane Events Involved in Autophagy

<div><p>(A) Upon induction of autophagy, a membrane sac called the isolation membrane (IM) forms and engulfs portions of the cytoplasm. Sealing of its edges gives rise to the double-membrane bound autophagosome. Fusion of the outer membrane with a lysosome results in formation of an autolysosome, in which the inner autophagosomal membrane and its contents are degraded.</p> <p>(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, whereas the contents of ALs show various stages of degradation.</p> <p>(C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively. Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).</p></div

Possible Membrane Sources of the IM

<p>Some of the possible scenarios for IM formation are illustrated here. According to the maturation model, upon induction of autophagy, membrane may be directly derived from the ER by folding (A), or in the form of vesicular transport (B). In the assembly model, membrane may be assembled de novo at the site of IM formation, originating from nonvesicular transport, such as micellar, as shown in (C), or local synthesis (D). See text for further details.</p

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

<div><p>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.</p> </div

Endogenous p62 accumulates in Atg and S6K mutants.

<p>A. p62 immunostaining detects small dots scattered throughout the cytoplasm in fat body cells of wild-type larvae. B. p62 aggregates appear bigger and more numerous in Atg1 null mutants, while loss of S6K (C) only increases the number, but not the size of p62 aggregates. D. Statistical evaluation of p62 puncta in fat bodies of various Atg mutants and in S6K null animals. * indicates a significant difference (p<0.05), ** indicates a very significant difference (p<0.01), based on two-tailed two-sample unequal Student’s t tests. Scalebar in panel A equals 30 µm for panels A–C. <i>Genotypes are: (A) w [1118]; (B) Atg1 [25]; (C) S6K[l–1].</i></p

The effect of Atg16, Atg18a and Pten RNAi lines on starvation-induced autophagy in L3 and L2 larval stages.

<p>Both RNAi lines for Atg16 show a similar block of mCherry-Atg8a puncta formation in L3 (compare A to C), while the size of these dots is reduced more efficiently by Atg16^HMS in L2 (compare B to D; see also panel E for statistics). Both RNAi lines for Atg18a strongly inhibit mCherry-Atg8a dot formation in L3 (compare F to H), while Atg18a^JF shows a more complete block in L2, reducing both the size and number of puncta (compare G to I; see also panel J for statistics). Both RNAi lines for Pten strongly inhibit mCherry-Atg8a dot formation in L3 (compare K to M), while Pten^JF shows a more complete block in L2, again reducing both the size and number of puncta (compare L to N; see also panel O for statistics). * indicates a significant difference (p<0.05), ** indicates a very significant difference (p<0.01), based on two-tailed two-sample unequal Student’st tests in panels E, J, O. Scalebar in panel A equals 30 µm for panels A, C, F, H, K, M, and scalebar in panel B equals 30 µm for panels B, D, G, I, L, N. <i>Genotypes are: (A, B) hsFlp; UAS-Dcr2/Atg16[KK105993]; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/+; (C, D) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/Atg16[HMS01347]; (F, G) hsFlp; UAS-Dcr2/Atg18a[KK105366]; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/+; (H, I) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/Atg18a[JF02898]; (K, L) hsFlp; UAS-Dcr2/Pten[KK101475]; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/+; (M, N) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/Pten[JF01987].</i></p

Loss of known autophagy regulators enhances p62 puncta formation cell-autonomously.

<p>Knockdown of Atg1 (A) or Tsc2 (B) increases p62 aggregate formation, while silencing of RpS8 results in a slight reduction of p62 dot number (C). Panel D shows statistical evaluation of the effect of RNAi and overexpression lines on p62 accumulation. Overexpressed p62-GFP forms multiple aggregates in control and Atg1 RNAi cells (panels E and F, respectively). Exposure times are indicated in the top right corner for panels E and F. Statistical evaluation of p62-GFP aggregate size and number in various RNAi and overexpression lines reveals changes that are difficult to interpret (G), but p62-GFP levels inferred from exposure times during image acquisition are qualitatively similar to data obtained with anti-p62 immunostaining (compare H to D). * indicates a significant difference (p<0.05), ** indicates a very significant difference (p<0.01), based on two-tailed two-sample unequal Student’s t tests in panels D, G, H. Scalebar in panel A equals 30 µm for panels A–C and E–F. <i>Genotypes are: (A) hsFlp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/Atg1[JF02273]; (B) hsFlp; UAS-Dcr2/TSC2[KK103417]; Act>CD2>Gal4, UAS-GFPnls/+; (C) hsFlp; UAS-Dcr2/RpS8[KK106835]; Act>CD2>Gal4, UAS-GFPnls/+; (E) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/+ (F) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/Atg1[JF02273].</i></p

p62 levels in Atg mutants, p62 RNAi and p62 overexpression cells.

<p>A. Western blot analysis shows p62 accumulation in Atg8a and Atg7 mutant heads and in Atg8a mutant larvae. RNAi knockdowns of p62 (i1, i2) greatly decrease endogenous protein levels, while overexpression of p62-GFP increases endogenous p62 levels in addition to the appearance of the 130 kDa extra band corresponding to the tagged protein. Numbers refer to p62 protein level relative to tubulin loading control for each sample. B-D. RNAi knockdown of p62 in fat body cell clones (marked by expression of Lamp-GFP) strongly decreases p62 puncta formation. E. Expression of p62-GFP in cell clones increases aggregate formation. Arrowhead in E’ indicates a large aggregate in a p62-GFP expressing cell, arrow marks an endogenous p62 dot in a control cell. F. Statistical evaluation of the number and size of p62 dots for samples in panels B–E. ** indicates a very significant difference (p<0.01), based on two-tailed two-sample unequal Student’s t tests. Scalebar in panel B equals 30 µm for panels B–E. <i>Genotypes are: (A) lane 1: w [1118], lane 2: Atg7[d77]/Atg7[d14], lane 3: Atg8a[d4], lane 4: UbiGal4/+; p62[HMS00551]/+, lane 5: UbiGal4/+; p62[HMS00938]/+, lane 6: w [1118], lane 7: Atg8a[d4], lane 8: cgGal4/UAS-p62-GFP; (B) hs-Flp; UAS-LampGFP/p62[KK108193]; Act>CD2>Gal4, UAS-Dcr2/+; (C) hs-Flp; UAS-LampGFP/+; Act>CD2>Gal4, UAS-Dcr2/p62[HMS00551]; (D) hs-Flp; UAS-LampGFP/+; Act>CD2>Gal4, UAS-Dcr2/p62[HMS00938]; (E) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4,UAS-Dcr2/+.</i></p

The effect of p62 and Atg8a reporters on autophagy phenotypes.

<p>A. Coexpression of p62-GFP and mCherry-Atg8a results in formation of aggregates containing both tagged proteins in fed animals, potentially suggesting enhanced autophagy. B. Expression of p62-GFP fails to induce Lysotracker puncta in fed animals. C. p62-GFP expression leads to the appearance of protein aggregates in ultrastructural images of fat body cells (asterisks), but no autophagic structures are seen in the cytoplasm. Inset shows an enlarged aggregate to illustrate that these inclusion bodies are not membrane-bound. D. Western blot analysis using anti-GFP antibodies of larval extracts expressing mCherry-Atg8a and p62-GFP in the fat body. No generation of free GFP by autolysosomal degradation is seen in well-fed control larvae, it is only induced by a 4-hour starvation or in the wandering stage. Silencing of Atg1 or expression of dominant-negative Vps34 inhibits autophagy-mediated conversion of p62-GFP to free GFP. E, F. Expression of dominant-negative Atg4 blocks starvation-induced Lysotracker puncta formation, but it has no effect on overexpressed mCherry-Atg8a dots. G. Ultrastructural analysis of starved fat bodies reveal small autophagosomes (marked by arrowheads in G’ and G’), but no autolysosomes are seen. Coexpression of GFP-Atg8a with dominant-negative Atg4 rescues this inhibition: numerous autolysosomes (arrow) and autophagosomes (arrowheads) are seen in H, H’. Scalebar in panel A equals 30 µm for panels A, B, E, F and scalebars in panels C, G, H equal 2 µm. <i>Genotypes are: (A) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/r4-mCherry-Atg8a; (B, C) hsFlp; UAS-p62-GFP/+; Act>CD2>Gal4, UAS-Dcr2/+; (D) lanes 1, 2, 3: cgGal4, UAS-p62-GFP/+; Atg1[JF02273]/+, lanes 4, 5, : cgGal4, UAS-Vps34[KD]/UAS-p62-GFP, lanes 6, 7, 8, : cgGal4, UAS-p62-GFP/+; (E, G) hs-Flp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls/UAS-Atg4[C98A]; (F, H) hs-Flp; UAS-Dcr2/+; Act>CD2>Gal4, UAS-GFPnls, r4-mCherry-Atg8a/UAS-Atg4[C98A].</i></p

All images depict live fat body tissue dissected from fed (C) or 4-h starved animals (all other panels)

(A) loss-of-function clones (marked by lack of GFP) fail to accumulate autolysosomes (labeled by punctate Lysotracker Red staining) after starvation. (B) Vps34-expressing cells (GFP positive) fail to accumulate punctate Lysotracker Red staining under starvation conditions. (C and D) Clonal expression of wild-type Vps34 (GFP-positive cells) does not induce autophagy under fed conditions (C) but increases the response to starvation (D). (E) loss-of-function clones (marked by lack of myrRFP) fail to accumulate autophagosomes (marked by punctate GFP-Atg8) in response to starvation. (F and G) Starvation-induced accumulation of GFP-Atg8a punctae is observed in controls (F) but not in animals expressing Vps34 (G). (H–K) TEM images reveal abundant autophagosomes (AP) and autolysosomes (AL) in control animals (H) but not mutants (I) nor animals expressing Vps34 (J). LD, lipid droplet. The graph in K shows autophagosome and autolysosome area ratios calculated from electron micrographs of five animals per genotype. Error bars show SD from the mean. P-values (Mann-Whitney test): control versus , AP, 1.45e-11 and AL, 3.9e-9; and control versus Vps34, AP, 2.9e-11 and AL, 1.5 e-11. Bars: (A–G) 10 μm; (H–J) 1 μm. Genotypes: (A) , (B) , (C and D) , (E) , (F) , (G) , (H) , (I) , (J) , and (K) as in H–J.<p><b>Copyright information:</b></p><p>Taken from "The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in "</p><p></p><p>The Journal of Cell Biology 2008;181(4):655-666.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386105.</p><p></p

(A) Mutation of the ESCRT-II component Vps28 (mutant clone marked by lack of GFP) disrupts formation of Lysotracker Red punctae in response to 4-h starvation

Bar, 10 μm. (B–D) GFP-Atg8a–marked autophagosomes accumulate in clones of mutant (B) and double mutant (D) cells but not in mutant cells (C). Mutant clones are marked by lack of myrRFP. (E–G) Mosaic eye imaginal discs showing accumulation of ubiquitin-positive punctae in (E) and (G) mutant clones but not in mutant clones (F). Lack of GFP marks mutant clones. Bar, 10 μm (B–G). Genotypes: (A) , (B) , (C) , (D) , (E) , (F) , and (G) .<p><b>Copyright information:</b></p><p>Taken from "The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in "</p><p></p><p>The Journal of Cell Biology 2008;181(4):655-666.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386105.</p><p></p