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Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome.

Tumbarello DA, Waxse BJ, Arden SD, Bright NA, Kendrick-Jones J, Buss F - Nat. Cell Biol. (2012)

Bottom Line: Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy.We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion.We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK. dat39@cam.ac.uk

ABSTRACT
Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

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The myosin VI binding partners, T6BP, NDP52, and optineurin, colocalise with myosin VI on autophagosomes and are required for autophagosome biogenesis(a) Immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM Bafilomycin A1 for 2 hours. Immunolabelling was performed against the indicated endogenous proteins, optineurin, T6BP, and NDP52 (red), and LC3 (green). Boxed regions provide higher magnification. From confocal images, a Pearson’s coefficient was calculated to estimate the degree of colocalisation of the different autophagy adaptors with LC3. As a negative control, a Pearson’s coefficient was calculated after rotation of one color image by 90 degrees. Results represent >20 cells from n=2 independent experiments. Scale bar, 20 μm. (b) Stable cherry-LC3 expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 hours to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of colocalisation appear white in the merged three-color images and boxed regions provide areas of higher magnification. Scale bar, 20 μm. A Pearson’s coefficient was calculated for the degree of colocalisation between the 3 colors. As a negative control, a Pearson’s coefficient was calculated after rotating one color image by 90 degrees. Results represent >10 cells from n=2 independent experiments. (c) Hela cells were either mock transfected or cotransfected with siRNA targeted against T6BP, NDP52, and optineurin (TNO). Cells were either left untreated or treated with100 nM Bafilomycin A1 (BfnA1) for 4 hours. Western blot analysis was performed against the indicated proteins. (d) Quantitation of LC3-II intensity (+/− s.d.) was performed from Western blot data of TNO siRNA treated Hela cells. The results represent n=3 independent experiments. **p<0.01, ***p<0.001 (e) Immunofluorescence microscopy of RFP-GFP-LC3 expressing Hela cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. Results are calculated from >100 cells from n=2 independent experiments represented as the Pearson’s coefficient of GFP/RFP signal overlap.
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Figure 5: The myosin VI binding partners, T6BP, NDP52, and optineurin, colocalise with myosin VI on autophagosomes and are required for autophagosome biogenesis(a) Immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM Bafilomycin A1 for 2 hours. Immunolabelling was performed against the indicated endogenous proteins, optineurin, T6BP, and NDP52 (red), and LC3 (green). Boxed regions provide higher magnification. From confocal images, a Pearson’s coefficient was calculated to estimate the degree of colocalisation of the different autophagy adaptors with LC3. As a negative control, a Pearson’s coefficient was calculated after rotation of one color image by 90 degrees. Results represent >20 cells from n=2 independent experiments. Scale bar, 20 μm. (b) Stable cherry-LC3 expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 hours to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of colocalisation appear white in the merged three-color images and boxed regions provide areas of higher magnification. Scale bar, 20 μm. A Pearson’s coefficient was calculated for the degree of colocalisation between the 3 colors. As a negative control, a Pearson’s coefficient was calculated after rotating one color image by 90 degrees. Results represent >10 cells from n=2 independent experiments. (c) Hela cells were either mock transfected or cotransfected with siRNA targeted against T6BP, NDP52, and optineurin (TNO). Cells were either left untreated or treated with100 nM Bafilomycin A1 (BfnA1) for 4 hours. Western blot analysis was performed against the indicated proteins. (d) Quantitation of LC3-II intensity (+/− s.d.) was performed from Western blot data of TNO siRNA treated Hela cells. The results represent n=3 independent experiments. **p<0.01, ***p<0.001 (e) Immunofluorescence microscopy of RFP-GFP-LC3 expressing Hela cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. Results are calculated from >100 cells from n=2 independent experiments represented as the Pearson’s coefficient of GFP/RFP signal overlap.

Mentions: The myosin VI binding partners, optineurin, NDP52 and T6BP all contain an ubiquitin-binding domain (UBD) and a LC3 interaction region (LIR), which are typically found in autophagy receptors such as p62 and NBR122. Since previous reports demonstrated a role for NDP52 and optineurin in autophagy-dependent clearance of Salmonella from mammalian cells7,8, we tested whether the function of these autophagy receptors is restricted to pathogen clearance or whether they have a general role in autophagy. Endogenous T6BP, NDP52 and optineurin are present on LC3-positive autophagosomes under basal autophagy conditions in the presence of BafilomycinA1 (Figure 5a). Interestingly, whereas localisation of NDP52 and T6BP to autophagosomes does not require their C-terminal zinc-finger motifs that mediate ubiquitin-binding, the ubiquitin-binding motif of optineurin, within the myosin VI interaction domain, disrupts its localisation to autophagosomes (Supplementary Figure S5a).


Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome.

Tumbarello DA, Waxse BJ, Arden SD, Bright NA, Kendrick-Jones J, Buss F - Nat. Cell Biol. (2012)

The myosin VI binding partners, T6BP, NDP52, and optineurin, colocalise with myosin VI on autophagosomes and are required for autophagosome biogenesis(a) Immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM Bafilomycin A1 for 2 hours. Immunolabelling was performed against the indicated endogenous proteins, optineurin, T6BP, and NDP52 (red), and LC3 (green). Boxed regions provide higher magnification. From confocal images, a Pearson’s coefficient was calculated to estimate the degree of colocalisation of the different autophagy adaptors with LC3. As a negative control, a Pearson’s coefficient was calculated after rotation of one color image by 90 degrees. Results represent >20 cells from n=2 independent experiments. Scale bar, 20 μm. (b) Stable cherry-LC3 expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 hours to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of colocalisation appear white in the merged three-color images and boxed regions provide areas of higher magnification. Scale bar, 20 μm. A Pearson’s coefficient was calculated for the degree of colocalisation between the 3 colors. As a negative control, a Pearson’s coefficient was calculated after rotating one color image by 90 degrees. Results represent >10 cells from n=2 independent experiments. (c) Hela cells were either mock transfected or cotransfected with siRNA targeted against T6BP, NDP52, and optineurin (TNO). Cells were either left untreated or treated with100 nM Bafilomycin A1 (BfnA1) for 4 hours. Western blot analysis was performed against the indicated proteins. (d) Quantitation of LC3-II intensity (+/− s.d.) was performed from Western blot data of TNO siRNA treated Hela cells. The results represent n=3 independent experiments. **p<0.01, ***p<0.001 (e) Immunofluorescence microscopy of RFP-GFP-LC3 expressing Hela cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. Results are calculated from >100 cells from n=2 independent experiments represented as the Pearson’s coefficient of GFP/RFP signal overlap.
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Figure 5: The myosin VI binding partners, T6BP, NDP52, and optineurin, colocalise with myosin VI on autophagosomes and are required for autophagosome biogenesis(a) Immunofluorescence microscopy was performed on RPE cells following treatment with 100 nM Bafilomycin A1 for 2 hours. Immunolabelling was performed against the indicated endogenous proteins, optineurin, T6BP, and NDP52 (red), and LC3 (green). Boxed regions provide higher magnification. From confocal images, a Pearson’s coefficient was calculated to estimate the degree of colocalisation of the different autophagy adaptors with LC3. As a negative control, a Pearson’s coefficient was calculated after rotation of one color image by 90 degrees. Results represent >20 cells from n=2 independent experiments. Scale bar, 20 μm. (b) Stable cherry-LC3 expressing RPE cells were transiently transfected with GFP-myosin VI, treated with Torin1 for 3 hours to induce autophagy, and subsequently processed for confocal immunofluorescence microscopy. Areas of colocalisation appear white in the merged three-color images and boxed regions provide areas of higher magnification. Scale bar, 20 μm. A Pearson’s coefficient was calculated for the degree of colocalisation between the 3 colors. As a negative control, a Pearson’s coefficient was calculated after rotating one color image by 90 degrees. Results represent >10 cells from n=2 independent experiments. (c) Hela cells were either mock transfected or cotransfected with siRNA targeted against T6BP, NDP52, and optineurin (TNO). Cells were either left untreated or treated with100 nM Bafilomycin A1 (BfnA1) for 4 hours. Western blot analysis was performed against the indicated proteins. (d) Quantitation of LC3-II intensity (+/− s.d.) was performed from Western blot data of TNO siRNA treated Hela cells. The results represent n=3 independent experiments. **p<0.01, ***p<0.001 (e) Immunofluorescence microscopy of RFP-GFP-LC3 expressing Hela cells transfected with TNO siRNA was performed and confocal images were evaluated for the correlation in GFP and RFP punctae signal overlap using the Image JACoP plugin. Results are calculated from >100 cells from n=2 independent experiments represented as the Pearson’s coefficient of GFP/RFP signal overlap.
Mentions: The myosin VI binding partners, optineurin, NDP52 and T6BP all contain an ubiquitin-binding domain (UBD) and a LC3 interaction region (LIR), which are typically found in autophagy receptors such as p62 and NBR122. Since previous reports demonstrated a role for NDP52 and optineurin in autophagy-dependent clearance of Salmonella from mammalian cells7,8, we tested whether the function of these autophagy receptors is restricted to pathogen clearance or whether they have a general role in autophagy. Endogenous T6BP, NDP52 and optineurin are present on LC3-positive autophagosomes under basal autophagy conditions in the presence of BafilomycinA1 (Figure 5a). Interestingly, whereas localisation of NDP52 and T6BP to autophagosomes does not require their C-terminal zinc-finger motifs that mediate ubiquitin-binding, the ubiquitin-binding motif of optineurin, within the myosin VI interaction domain, disrupts its localisation to autophagosomes (Supplementary Figure S5a).

Bottom Line: Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy.We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion.We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK. dat39@cam.ac.uk

ABSTRACT
Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitylated cargoes are recognized by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content; however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes, and demonstrate that loss of myosin VI and Tom1 reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin, thereby promoting autophagosome maturation and thus driving fusion with lysosomes.

Show MeSH