Uba1 functions in Atg7- and Atg3-independent autophagy

Abstract

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.

Figure 1

Atg18 and Atg2 are required for programmed cell size reduction in the Drosophila midgut. (a) Representative differential interference contrast (DIC) microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3^rd), late third instar larval (Late 3^rd) and at puparium formation (white prepupal, WPP) stages. (b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown. (c) Midguts from control Atg18^KG03090/wild-type (Atg18/+), n =14, and Atg18^KG03090/Df(3L)⁶¹¹² mutant (Atg18/Df), n = 11, animals at puparium formation analyzed by DIC microscopy. Representative images are shown. (d) Wild-type, control (Atg18^KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm²) at indicated stages, n = 10 animal intestines/genotype with 5 cells measured/intestine/stage. (e) DIC images of midgut cells from Atg2^EP3697/wild-type control (Atg2/+) and Atg2^EP3697/Df(3L)⁶⁰⁹¹ mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm²) from e, n = 12 (Atg2/+) and n = 7 (Atg2/Df) animal intestines/genotype with 5 cells measured/intestine. (g-j) Representative TEM images of intestine cells 2 hours after puparium formation. (g,i) Control Atg18^KG03090/wild-type (Atg18/+, g) and Atg2^EP3697/wild-type control (Atg2/+, i) cells with an enlarged image showing a double membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18^KG03090/Df(3L)⁶¹¹² mutant (Atg18/Df, h) cell and Atg2^EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± standard deviation (s.d.). Scale bars represent 20 μm (a-c and e) and 1 μm (g-j).

Figure 2

Programmed size reduction is cell autonomous and requires Atg18 and Atg1. (a) Midguts dissected from animals expressing Atg18^IR specifically in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (b) Quantification (μm²) from a, n = 12 animal intestines/genotype with 1-5 cells measured/intestine. (c) Midguts expressing mCherry-Atg8a in all cells, and expressing Atg18^IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown. (d) Midguts dissected from animals at puparium formation that contain an Atg1^Δ3D loss-of-function mutant cell clone (lacking GFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1^Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm²) from d, n = 7 (+/+), n = 8 (Δ3D/+), and n = 8 (Δ3D/Δ3D) animal intestines/genotype with 1-5 cells measured/intestine. (f) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Atg1^IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown. (g-j) Representative cut views from 3D reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early 3^rd instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm³) from g-j, n = 5 animal intestines/genotype with 1-5 cells measured/intestine/stage. Black columns: early 3^rd instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d.. N.S.: no significance. Scale bars represent 20 μm.

Figure 3

Autophagy is necessary and sufficient for cell size reduction. (a) Midguts dissected from animals at puparium formation that contain Vps34^Δm22 loss-of-function mutant cell clones (lacking GFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34^Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm²) from a, n = 14 animal intestines/genotype with 1-5 cells measured/intestine. (c) Midguts dissected from animals expressing Atg8a^IR specifically in GFP-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (d) Cell size quantification (μm²) from c, n = 10 animal intestines/genotype with 1-5 cells measured/intestine. (e) Midguts dissected from animals expressing Atg12^IR specifically in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (f) Cell size quantification (μm²) from e, n = 14 animal intestines/genotype with 1-5 cells measured/intestine. (g-i) Midguts dissected from early third instar larvae that mis-express Atg1^GS10797 (Atg1) (g, GFP in nucleus and cytoplasm), both Atg1 + Atg8a^IR (h), or both Atg1 + Atg7^IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g’ and i’) DIC images of midguts expressing mCherry-Atg8a in all cells, Atg1 (g’), and Atg1 + Atg7^IR (i’) in GFP-marked clones of cells. mCherry-Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d.. Scale bars represent 20 μm.

Figure 4

Atg7 is not required for programmed cell size reduction and autophagy. (a) Midguts dissected from animals at puparium formation that contain Atg7^d4 loss-of-function mutant cell clones (lacking GFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7^d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm²) from a, n = 8 animal intestines/genotype with 1-5 cells measured/intestine. (c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7^d4 loss-of-function clone cells (lacking GFP) at 2h after puparium formation. Representative images are shown. (d) Gastric caeca expressing GFP-Atg8a in all cells from control (Atg7^d30/d14) and Atg7 mutant (Atg7^d77/d14) animals. Representative images are shown. (e) Quantification of GFP-Atg8a puncta number from d, n = 31 animal intestines/genotype with puncta quantified in image field/intestine. (f) Midguts expressing GFP-Atg5 in in enterocytes (larger nuclei) with an Atg7^d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown. (g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g) Control (Atg7^d30/d14) and (h) Atg7 mutant (Atg7^d77/d14) cells both contain autophagic structures. (h) Enlarged Atg7 mutant cell image of a double membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d.. N.S.: no significance. Scale bars represent 20 μm (a,c,d, and f) and 1 μm (g,h).

Figure 5

Autophagy is required for clearance of mitochondria. (a) Quantification of mitochondria numbers from Atg18 control (Atg18^KG03090/wild-type) and mutant (Atg18^KG03090/Df(3L)⁶¹¹²) or Atg7 control (Atg7^d30/d14) and mutant (Atg7^d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm² regions per cell from 2 cells per animal from at least 3 different animals/genotype. (b, c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labeled mitochondria in all cells and contain an Atg1^Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d.. N.S.: no significance. Scale bars represent 20 μm.

Figure 6

Uba1 is required for midgut cell programmed size reduction and autophagy. (a) Midguts dissected from animals expressing Uba1^IR specifically in GFP-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (b) Cell size quantification (μm²) from a, n = 16 animal intestines/genotype with 1-5 cells measured/intestine. (c) Midguts dissected from animals at puparium formation that contain Uba1^H33 loss-of-function mutant cell clones (lacking RFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1^H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm²) from c, n = 8 (+/+), n = 11 (H33/+), and n =11 (H33/H33) animal intestines/genotype with 1-5 cells measured/intestine. (e) Midguts dissected from animals at puparium formation that contain Uba1^H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analyzed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1^H33 loss-of-function clone cell (lacking RFP) at puparium formation analyzed by fluorescence microscopy. Representative images are shown. (g) Midguts dissected from animals at puparium formation that contain Uba1^H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analyzed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (h) Midguts dissected from animals expressing Dts7, a dominant temperature sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (i) Cell size quantification (μm²) from h, n = 6 animal intestines/genotype with 1-5 cells measured/intestine. Quantification is shown as mean ± s.d.. N.S.: no significance. Scale bars represent 20 μm.

Figure 7

Role of Uba1 in midgut autophagy. (a and b) E1 charging assay for Uba1 and Atg7 with either Atg8a (a) or Ub (b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Molecular weight ladders are indicated. Representative images are shown. Molecular weights: Flag-Uba1 135 kDa, Flag-Atg7 81 kDa, His-Atg8a 29kDa, Ub 9 kDa. (c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7^d77/d14) and Atg8 (Atg8^KG07569) mutant blotted with anti-Atg8 and anti-Tubulin, n = 3 independent biological experiments. Representative images are shown. (d) Midguts from control Atg3¹⁰/wild-type (Atg3/+), and Atg3¹⁰/Df(3L)cat mutant (Atg3/Df) animals at puparium formation analyzed by DIC microscopy. Representative images are shown. (e) Cell size quantification (μm²) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines/genotype with 5 cells measured/intestine. (f) Midguts dissected from early 3^rd instar larvae that mis-express Atg1^GS10797 (Atg1) and Atg3^IR only in the DsRed-marked cell clone and analyzed by fluorescence and DIC microscopy. Representative images are shown. (g) Midguts dissected from animals expressing Atg4^DN specifically in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (h) Cell size quantification (μm²) from g, n = 10 animal intestines/genotype with 1-5 cells measured/intestine. (i) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Ub^IR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown. (j) Midguts dissected from animals expressing Ub^IR specifically in GFP-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (k) Cell size quantification (μm²) from j, n = 12 animal intestines/genotype with 1-5 cells measured/intestine. (l,m) Midguts dissected from early third instar larvae that contain Atg1^GS10797 (Atg1) mis-expression in either a wild-type GFP positive cell clone (l) or a Uba1^H33 loss-of-function MARCM mutant cell clone (GFP-positive) (m) and analyzed by fluorescence and DIC microscopy. Control wild-type and heterozygous Uba1^H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm²) from l and m, n = 22 (control), n = 16 (Atg1), and n =6 (Atg1 + Uba1^H33) animal intestines/genotype with 1-5 cells measured/intestine. Quantification of control cells (l,m) were pooled since they are genetically identical. Quantification is shown as mean ± s.d.. N.S.: no significance. Scale bars represent 20 μm.

Figure 8

Uba1 is required for clearance of mitochondria. (a, b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labeled mitochondria in enterocytes (larger nuclei) and contain Uba1^H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. (c) Midguts dissected at puparium formation that express Uba1^IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown. (d-f) Representative immuno-TEM images of Uba1^IR clone cells at puparium formation. Uba1^IR-expressing cells possess gold particles, while control cells lack gold particles (d). Control cells possess numerous autolysosomes in the cytoplasm (arrowheads) and few mitochondria (asterisks), while Uba1^IR-expressing cells possess numerous mitochondria and few autophagic structures. Scale bars represent 20 μm (a-c) and 1 μm (d-f).