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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 targets to autophagosomes via the RRL motif in the cargo binding tail domain(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ Calmodulin-binding motif, and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. Additionally, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 hours to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin-extraction. Arrowheads indicate areas of colocalisation. Scale bar, 20 μm. (c) A Pearson’s coefficient was calculated based on the degree of colocalisation between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence images. Graph represents data from more than 20 transfected cells from n=2 independent experiments.
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Figure 4: Myosin VI targets to autophagosomes via the RRL motif in the cargo binding tail domain(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ Calmodulin-binding motif, and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. Additionally, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 hours to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin-extraction. Arrowheads indicate areas of colocalisation. Scale bar, 20 μm. (c) A Pearson’s coefficient was calculated based on the degree of colocalisation between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence images. Graph represents data from more than 20 transfected cells from n=2 independent experiments.

Mentions: Two primary protein-protein interaction regions with either a WWY or RRL motif are present in the C-terminal cargo-binding tail region of myosin VI, which also contains a ubiquitin-binding domain (UBD)21 (Figure 4a). To identify how myosin VI is recruited to autophagosomes, GFP-myosin VI tail constructs, containing either a WWY to WLY substitution (ΔWWY), RRL to AAA substitution (ΔRRL), or A1013G point mutation in the UBD, were transiently expressed in RPE cells stably expressing cherry-LC3. To visualize the precise intracellular localisation of the GFP-myosin VI tails, the cells were briefly extracted with saponin to remove the cytosolic pool of the GFP-myosin VI tail before fixation. The wild-type myosin VI tail and the tail mutants with the ΔWWY and A1013G mutations show prominent colocalisation with LC3-positive autophagosomes (Figure 4b,c), which is significantly decreased without the RRL motif, which mediates interactions with T6BP, NDP52, and optineurin (Figure 4a,b,c).


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 targets to autophagosomes via the RRL motif in the cargo binding tail domain(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ Calmodulin-binding motif, and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. Additionally, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 hours to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin-extraction. Arrowheads indicate areas of colocalisation. Scale bar, 20 μm. (c) A Pearson’s coefficient was calculated based on the degree of colocalisation between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence images. Graph represents data from more than 20 transfected cells from n=2 independent experiments.
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Related In: Results  -  Collection

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Figure 4: Myosin VI targets to autophagosomes via the RRL motif in the cargo binding tail domain(a) Cartoon illustration of myosin VI domain structure. Myosin VI contains a catalytic motor domain, a unique insert (green), an IQ Calmodulin-binding motif, and a cargo-binding tail domain. The cargo-binding tail domain contains multiple protein interaction motifs (RRL and WWY) as well as regions for ubiquitin and PIP2 binding. Additionally, two alternative splicing events give rise to two insertions in the cargo-binding tail region. (b) RPE cells with stable expression of cherry-LC3 were transiently transfected with the GFP-myosin VI cargo-binding tail domain containing various mutations in the protein interaction (ΔWWY and ΔRRL) and ubiquitin-binding motifs (A1013G), followed by treatment with 250 nM Torin1 for 3 hours to induce autophagy. Immunofluorescence microscopy was performed either in the absence or presence of saponin-extraction. Arrowheads indicate areas of colocalisation. Scale bar, 20 μm. (c) A Pearson’s coefficient was calculated based on the degree of colocalisation between the different GFP-myosin VI mutant tails and cherry-LC3 from confocal immunofluorescence images. Graph represents data from more than 20 transfected cells from n=2 independent experiments.
Mentions: Two primary protein-protein interaction regions with either a WWY or RRL motif are present in the C-terminal cargo-binding tail region of myosin VI, which also contains a ubiquitin-binding domain (UBD)21 (Figure 4a). To identify how myosin VI is recruited to autophagosomes, GFP-myosin VI tail constructs, containing either a WWY to WLY substitution (ΔWWY), RRL to AAA substitution (ΔRRL), or A1013G point mutation in the UBD, were transiently expressed in RPE cells stably expressing cherry-LC3. To visualize the precise intracellular localisation of the GFP-myosin VI tails, the cells were briefly extracted with saponin to remove the cytosolic pool of the GFP-myosin VI tail before fixation. The wild-type myosin VI tail and the tail mutants with the ΔWWY and A1013G mutations show prominent colocalisation with LC3-positive autophagosomes (Figure 4b,c), which is significantly decreased without the RRL motif, which mediates interactions with T6BP, NDP52, and optineurin (Figure 4a,b,c).

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