EMBO/soda-nel huggingface dataset:
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 1

Clathrin‐mediated endocytosis is required for necrotic cell death in C. elegans. (A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d) mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses. (B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test). (C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia. (D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals. (E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide. (F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 2

Endosome formation is induced early during necrosis. (A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2::GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons of mec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis. (B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later. (C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 3

Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT‐1) or endophilin (UNC‐57) does not further suppress MEC‐4(d)‐induced necrosis in cad‐1 mutants with reduced cathepsin activity or with V‐ATPase dysfunction (B, C). Similarly, no synthetic reduction of necrotic cell death is observed in calpain‐deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 4

Endocytosis synergizes with autophagy to mediate necrotic cell death. (A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy. (B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 5

Intracellular trafficking by kinesins UNC‐116 and UNC‐104 is required for necrotic cell death. (A) Depletion of the kinesin 1 heavy chain UNC‐116 suppresses necrosis induced by both mec‐4(d) and deg‐3(d) toxic alleles. (B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, unc‐116 and unc‐104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test). (C) Percentage of unc‐116 animals that survive after treatment with sodium azide. (D) Depletion of the monomeric kinesin UNC‐104 protects neurons against MEC‐4(d)‐induced degeneration. (E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide. (F) Combined inactivation of endocytosis and kinesin‐mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC‐4(d)‐induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 6

Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC‐116) or the monomeric kinesin UNC‐104 does not significantly enhance suppression of neurodegeneration in aspartyl protease‐deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease‐deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).
Endocytosis and intracellular trafficking contribute to necrotic neurodegeneration in C. elegans (2011)
Figure 7

Kinesin‐mediated intracellular trafficking functions together with autophagy to facilitate necrotic cell death. Number of neuron corpses, at the L1 larval stage of development, per 100 animals carrying the neurotoxic mec‐4(d) allele in genetic backgrounds deficient for both intracellular trafficking and autophagy. (A) Kinesin 1 heavy chain (UNC‐116) deficiency. (B) Monomeric kinesin UNC‐104 deficiency. In both cases, no significant synthetic protection of neurons is observed in LGG‐1/LC3‐depleted animals with impaired autophagy. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 1

Screening of active members of the myotubularin family identifies Jumpy as a negative regulator of autophagy. (A) Domains and members of catalytically active myotubularins. Asterisks, Jumpy mutations found in patients with centronuclear myopathy. C330S, R336Q and Y462C, Jumpy mutants used in this study. (B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium. (C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm. (E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t‐test). Scale bars, 10 μm. (G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5–8). Inset shows Atg5 knock‐down by immunoblotting.
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 2

Jumpy blocks autophagic degradation. (A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments. (B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments). (C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3). (D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5). (E–M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments). (N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O). (P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 3

Jumpy localizes to autophagosomes. (A) Transient association of Jumpy WT with LC3+ organelles. Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP‐Jumpy WT and Cherry‐LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope. EBSS was added to the cells and z‐stacks collected at 3‐min intervals for a total of 45 min. The collected images were processed to generate a maximum projection (collapsing a 3D image into an x–y projection) for each time point. Arrows indicate colocalization of GFP‐Jumpy WT with LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP‐Jumpy WT colocalizing with Cherry‐LC3+‐positive puncta indicated by arrows. (B–I) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐G58K (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B–E) are shown at higher magnification in the corresponding panel below (F–I). Scale bars, 2 μm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow indicates colocalization between Jumpy CS and G58K but not LC3.
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 4

Jumpy localizes to autophagic isolation membranes. (A–H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A–D) are shown at higher magnification in the corresponding panel below (E–H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3. (I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J). (K–R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K–N) are shown at higher magnification in the corresponding panel below (O–R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3. (S–ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S–V) are shown at higher magnification in the corresponding panel below (W–ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 5

Jumpy siRNA increases recruitment of PI3P‐binding Atg factor WIPI‐1 (Atg18) to autophagic membranes. (A–C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets. (D–I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F–H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E–H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 6

Jumpy colocalizes with and regulates Atg9 distribution to autophagic organelles. (A–T) C2C12 cells were transfected for 24 h with GFP, GFP‐Jumpy WT or GFP‐Jumpy C330S (Jumpy CS) and Tomato‐LC3 (LC3) and HA‐Atg9, fixed and immunostained with anti‐HA. GFP (I), Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A–D) are shown at higher magnification in the corresponding panel below (E–H). White arrows indicate colocalization between Jumpy CS, LC3 and Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9. White boxes (I–T) show LC3 puncta location. Scale bars, 5 μm. (U) Percentage of LC3 puncta colocalizing with Atg9. n, number of puncta counted.
Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy (2009)
Figure 7

Jumpy R336Q mutant associated with centronuclear myopathy is defective in inhibiting autophagy. (A–K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant. (L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 1

Mitochondrial respiratory deficiency impairs ATG8 induction and autophagic flux. (A) Wild‐type and rho0 cells harbouring prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels) were exposed to amino‐acid or nitrogen starvation (‐N) medium supplemented with indicated carbon sources. Cells were analysed at indicated time points by whole cell extraction and western blot analysis using α‐GFP and α‐Cdc11 antibodies. Quantification of ATG8 induction is shown in the lower left panel. Total GFP signals (GFP‐Atg8 and free GFP) were quantified and normalized to Cdc11 signals. Normalized values at 0 h were set as one and relative changes are shown after 6 h starvation. Quantification of autophagic flux is shown in the lower right panel as ratio of free GFP to total GFP signals (GFP‐Atg8 and free GFP) after 6 h starvation. The means and s.d. of four (n=4) independent experiments are indicated. (B) Fluorescence microscopical analysis. Wild‐type, rho0, and Δatg7 cells expressing prATG8‐GFP‐ATG8 were grown as described in (A) and exposed to amino‐acid (left panel, galactose) or nitrogen starvation (right panel, glucose (‐N)) for 6 h. Vacuoles were visualized by over night FM4‐64 (1 μM) staining (red). Arrowhead indicates a punctate GFP‐Atg8 structure. Transmission and fluorescence light microscopy images were superimposed to visualize cellular boundaries. Cellular localization of GFP signal was analysed in at least 150 cells (n⩾150) for each strain and condition. Scale bars represent 1.5 μm.
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 2

Autophagic response in cells with compromised respiratory chain complex III, IV, or V activity during amino‐acid starvation. Wild‐type, rho0, Δatg7 cells and mutants that are selectively inhibited in the biogenesis of respiratory chain complex III (CIII: Δcbs1), complex IV (CIV: Δmss51, Δpet111, Δpet122), or the F1Fo‐ATP synthase (complex V; CV: Δatp10) were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. All strains expressed prATG8‐GFP‐ATG8 (upper panels) or prATG8‐GFP (lower panels). Samples were analysed as described in Figure 1A. The means and s.d. of five (n=5) independent experiments are indicated.
Mitochondria regulate autophagy by conserved signalling pathways (2011)
Figure 3

ATP level and mitochondrial membrane potential during amino‐acid starvation. (A, B) Wild‐type, rho0, and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate (A) or galactose (B). When indicated, wild‐type cells were exposed to antimycin A (AA) or oligomycin (O) during the amino‐acid starvation period. ATP and protein from total cells (upper panels) or isolated mitochondria (lower panels) were determined at indicated time points. (C) Wild‐type and Δatg7 cells were exposed to amino‐acid starvation medium supplemented with acetate or galactose for 3 h. Wild‐type cells were exposed to antimycin A (AA), oligomycin (O), or CCCP (50 μM) during the amino‐acid starvation period when indicated. Cells were treated with the mitochondrial membrane potential‐dependent dye DiOC6(3) and examined by fluorescence microscopy. Average pixel intensities for at least 10 mitochondrial tubules (n=10) in 5 representative cells were determined for each condition. Values for wild‐type mitochondria in the presence of acetate were defined as 100% and relative values were calculated accordingly. The means and s.d. are shown.