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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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Myosin VI mediates delivery of endocytic cargo to autophagosomes(a) RPE cells with stable expression of GFP-LC3 following mock or myosin VI siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labeled with Hoechst. Inserts provide higher magnification of boxed regions and arrows highlight areas of complete overlapping colocalisation and arrowheads highlight adjacent localisation of Tom1 relative to LC3. Scale bar, 20 μm. Quantitation was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. Results are represented as the average number of Tom1-positive vesicles/cell categorised by their relative position to LC3 (+/− s.d.) (n=3). (b) GFP-LC3 expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labeled with Texas Red Dextran (red) for 4 hours, prior to chase into fresh media containing 1 μM MG132 for 2 hours. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labeled with Hoechst (blue). Insets represent higher magnification of boxed regions. Scale bar, 20 μm. (c) Mander’s overlap coefficients for the degree of LC3 signal colocalising with Dextran were calculated using Volocity software. Results represent >20 cells from n=3 independent experiments and are illustrated as a box and whisker plot. Box represents median, 25th and 75th percentiles and whiskers represent max and min. (d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-Red Dextran for 16 hours followed by a chase period of 4 hours. Cells were processed for immunoelectron microscopy and labeled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas Red-Dextran. Scale bar, 200 nm.
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Figure 8: Myosin VI mediates delivery of endocytic cargo to autophagosomes(a) RPE cells with stable expression of GFP-LC3 following mock or myosin VI siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labeled with Hoechst. Inserts provide higher magnification of boxed regions and arrows highlight areas of complete overlapping colocalisation and arrowheads highlight adjacent localisation of Tom1 relative to LC3. Scale bar, 20 μm. Quantitation was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. Results are represented as the average number of Tom1-positive vesicles/cell categorised by their relative position to LC3 (+/− s.d.) (n=3). (b) GFP-LC3 expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labeled with Texas Red Dextran (red) for 4 hours, prior to chase into fresh media containing 1 μM MG132 for 2 hours. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labeled with Hoechst (blue). Insets represent higher magnification of boxed regions. Scale bar, 20 μm. (c) Mander’s overlap coefficients for the degree of LC3 signal colocalising with Dextran were calculated using Volocity software. Results represent >20 cells from n=3 independent experiments and are illustrated as a box and whisker plot. Box represents median, 25th and 75th percentiles and whiskers represent max and min. (d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-Red Dextran for 16 hours followed by a chase period of 4 hours. Cells were processed for immunoelectron microscopy and labeled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas Red-Dextran. Scale bar, 200 nm.

Mentions: Next, we determined whether Tom1 is required for autophagy. SiRNA-mediated knockdown of Tom1 leads to an accumulation of LC3-II (Figure 7a,b; Supplementary Figure S8a), and an increase in the number of LC3-positive autophagosomes and p62-positive punctae visualised by immunofluorescence microscopy (Figure 7c,d; Supplementary Figure S8b), very similar to the phenotype observed in myosin VI knockdown cells (Figure 1a-d). In the Hela RFP-GFP-LC3 stable cell line, loss of Tom1 expression leads to an accumulation of LC3-positive, yellow autophagosomes that are Cathepsin D- and Lamp1-negative (Figure 7e,f; Supplementary Figure S2a). We also observed in Tom1 knockdown cells a defect in protein aggregate clearance and thus an increase in the number of cells with multiple p62 and LC3-positive HttQ72-GFP aggregates (Figure 7g; Supplementary Figure S3), phenocopying the knockdown of myosin VI (Figure 2d,e; Supplementary Figure S3). Given the very similar requirement for Tom1 and myosin VI during late stages of autophagy, we next tested whether Tom1 is recruited to autophagosomes and whether this localisation was myosin VI-dependent. As shown in figure 8a, endogenous Tom1 is present on LC3-positive autophagosomes. However, siRNA knockdown of myosin VI causes an increase in the number of Tom1-positive vesicles that are adjacent to, but not completely colocalising with LC3-positive autophagosomes, suggesting a defect in docking or fusion of these vesicles with autophagosomes (Figure 8a). In summary, myosin VI and Tom1 function together in the final stages of autophagy, since the loss of either protein leads to an accumulation of autophagosomes unable to mature to an autolysosome.


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)

Myosin VI mediates delivery of endocytic cargo to autophagosomes(a) RPE cells with stable expression of GFP-LC3 following mock or myosin VI siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labeled with Hoechst. Inserts provide higher magnification of boxed regions and arrows highlight areas of complete overlapping colocalisation and arrowheads highlight adjacent localisation of Tom1 relative to LC3. Scale bar, 20 μm. Quantitation was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. Results are represented as the average number of Tom1-positive vesicles/cell categorised by their relative position to LC3 (+/− s.d.) (n=3). (b) GFP-LC3 expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labeled with Texas Red Dextran (red) for 4 hours, prior to chase into fresh media containing 1 μM MG132 for 2 hours. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labeled with Hoechst (blue). Insets represent higher magnification of boxed regions. Scale bar, 20 μm. (c) Mander’s overlap coefficients for the degree of LC3 signal colocalising with Dextran were calculated using Volocity software. Results represent >20 cells from n=3 independent experiments and are illustrated as a box and whisker plot. Box represents median, 25th and 75th percentiles and whiskers represent max and min. (d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-Red Dextran for 16 hours followed by a chase period of 4 hours. Cells were processed for immunoelectron microscopy and labeled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas Red-Dextran. Scale bar, 200 nm.
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Figure 8: Myosin VI mediates delivery of endocytic cargo to autophagosomes(a) RPE cells with stable expression of GFP-LC3 following mock or myosin VI siRNA transfection were processed for confocal immunofluorescence microscopy and immunolabelled for Tom1/Tom1L2 (red). Nuclei (blue) were labeled with Hoechst. Inserts provide higher magnification of boxed regions and arrows highlight areas of complete overlapping colocalisation and arrowheads highlight adjacent localisation of Tom1 relative to LC3. Scale bar, 20 μm. Quantitation was performed on >75 cells for the relative position (adjacent or complete overlap) of Tom1-positive vesicles in relation to LC3-positive vesicles. Results are represented as the average number of Tom1-positive vesicles/cell categorised by their relative position to LC3 (+/− s.d.) (n=3). (b) GFP-LC3 expressing RPE cells were depleted of myosin VI by siRNA. Cells were pulse labeled with Texas Red Dextran (red) for 4 hours, prior to chase into fresh media containing 1 μM MG132 for 2 hours. Cells were processed for immunofluorescence microscopy and immunostained for GFP (green). Nuclei were labeled with Hoechst (blue). Insets represent higher magnification of boxed regions. Scale bar, 20 μm. (c) Mander’s overlap coefficients for the degree of LC3 signal colocalising with Dextran were calculated using Volocity software. Results represent >20 cells from n=3 independent experiments and are illustrated as a box and whisker plot. Box represents median, 25th and 75th percentiles and whiskers represent max and min. (d) RPE cells stably expressing GFP-LC3 were depleted of myosin VI and Tom1 by siRNA. Cells were pulse-labelled with Texas-Red Dextran for 16 hours followed by a chase period of 4 hours. Cells were processed for immunoelectron microscopy and labeled with 15 nm gold particles against GFP-LC3 and 5 nm gold particles against Texas Red-Dextran. Scale bar, 200 nm.
Mentions: Next, we determined whether Tom1 is required for autophagy. SiRNA-mediated knockdown of Tom1 leads to an accumulation of LC3-II (Figure 7a,b; Supplementary Figure S8a), and an increase in the number of LC3-positive autophagosomes and p62-positive punctae visualised by immunofluorescence microscopy (Figure 7c,d; Supplementary Figure S8b), very similar to the phenotype observed in myosin VI knockdown cells (Figure 1a-d). In the Hela RFP-GFP-LC3 stable cell line, loss of Tom1 expression leads to an accumulation of LC3-positive, yellow autophagosomes that are Cathepsin D- and Lamp1-negative (Figure 7e,f; Supplementary Figure S2a). We also observed in Tom1 knockdown cells a defect in protein aggregate clearance and thus an increase in the number of cells with multiple p62 and LC3-positive HttQ72-GFP aggregates (Figure 7g; Supplementary Figure S3), phenocopying the knockdown of myosin VI (Figure 2d,e; Supplementary Figure S3). Given the very similar requirement for Tom1 and myosin VI during late stages of autophagy, we next tested whether Tom1 is recruited to autophagosomes and whether this localisation was myosin VI-dependent. As shown in figure 8a, endogenous Tom1 is present on LC3-positive autophagosomes. However, siRNA knockdown of myosin VI causes an increase in the number of Tom1-positive vesicles that are adjacent to, but not completely colocalising with LC3-positive autophagosomes, suggesting a defect in docking or fusion of these vesicles with autophagosomes (Figure 8a). In summary, myosin VI and Tom1 function together in the final stages of autophagy, since the loss of either protein leads to an accumulation of autophagosomes unable to mature to an autolysosome.

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
Related in: MedlinePlus