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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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Tom1 interacts with myosin VI and is required for myosin VI localisation to endosomes(a) Myosin VI and Tom1/Tom1L2 coimmunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by Western blot with antibodies to Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. The mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n=2 independent experiments. VHS = Vps-27, Hrs and STAM, GAT = GGA and Tom1. (c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI (green) and endogenous Tom1/Tom1L2 (red) or Rab5 (red) colocalisation. Inserts provide higher magnification of boxed regions. Arrows indicate areas of colocalisation. Scale bar, 20 μm. (d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by Western blot analysis and immunofluorescence microscopy to evaluate GFP-myosin VI tail localisation. Cells were either fixed directly or saponin-extracted prior to fixation. Scale bar, 20 μm. Quantitation was performed on GFP-myosin VI tail stable expressing RPE cells transfected with mock, Tom1, or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labeled with Hoechst followed by quantitation of GFP-myosin VI tail punctae/cell using an automated Cellomics VTi microscope. More than 600 cells/group from n=3 independent experiments were analysed and represented as the mean number of GFP-myosin VI tail punctae/cell (+/− s.d.).
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Figure 6: Tom1 interacts with myosin VI and is required for myosin VI localisation to endosomes(a) Myosin VI and Tom1/Tom1L2 coimmunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by Western blot with antibodies to Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. The mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n=2 independent experiments. VHS = Vps-27, Hrs and STAM, GAT = GGA and Tom1. (c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI (green) and endogenous Tom1/Tom1L2 (red) or Rab5 (red) colocalisation. Inserts provide higher magnification of boxed regions. Arrows indicate areas of colocalisation. Scale bar, 20 μm. (d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by Western blot analysis and immunofluorescence microscopy to evaluate GFP-myosin VI tail localisation. Cells were either fixed directly or saponin-extracted prior to fixation. Scale bar, 20 μm. Quantitation was performed on GFP-myosin VI tail stable expressing RPE cells transfected with mock, Tom1, or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labeled with Hoechst followed by quantitation of GFP-myosin VI tail punctae/cell using an automated Cellomics VTi microscope. More than 600 cells/group from n=3 independent experiments were analysed and represented as the mean number of GFP-myosin VI tail punctae/cell (+/− s.d.).

Mentions: The Drosophila homologue of the Tom1 protein family, CG3529, which is an alternate endosomal sorting complex required for transport (ESCRT) class 0, was recently identified as a myosin VI binding partner23,24,25. Since the ESCRTs are required for autophagosome maturation26 , we tested whether Tom1 and myosin VI interact in mammalian cells and whether Tom1 is required for autophagy. Myosin VI and Tom1, as well as its very close homologue Tom1L2, interact in mammalian cells as they can be coimmunoprecipitated with myosin VI antibodies (Figure 6a). Using a mammalian two-hybrid assay9,10,27 we show that Tom1 binds to myosin VI via the WWY motif and not via the RRL motif, which binds optineurin, NDP52 or T6BP. The myosin VI binding site on Tom1 resides within the final 104 amino acids of the C-terminus (Figure 6b). Myosin VI and Tom1 colocalise on vesicles located along the cell periphery, that are positive for endosomal markers APPL1, GIPC, and Rab5 (Figure 6c; Supplementary Figure S6a,b). In the absence of Tom1 and Tom1L2, myosin VI is lost from endosomes (Figure 6d), and accumulates on F-actin structures throughout the cell (Supplementary Figure S7). A partial effect was observed following knockdown of the myosin VI binding partner GIPC; however, loss of T6BP, NDP52 or optineurin did not effect myosin VI localisation (Figure 6d; Supplementary Figure S6c). Thus, the Tom1 protein family is a newly identified class of myosin VI adaptor proteins that are important for targeting myosin VI to endocytic structures in mammalian cells.


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)

Tom1 interacts with myosin VI and is required for myosin VI localisation to endosomes(a) Myosin VI and Tom1/Tom1L2 coimmunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by Western blot with antibodies to Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. The mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n=2 independent experiments. VHS = Vps-27, Hrs and STAM, GAT = GGA and Tom1. (c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI (green) and endogenous Tom1/Tom1L2 (red) or Rab5 (red) colocalisation. Inserts provide higher magnification of boxed regions. Arrows indicate areas of colocalisation. Scale bar, 20 μm. (d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by Western blot analysis and immunofluorescence microscopy to evaluate GFP-myosin VI tail localisation. Cells were either fixed directly or saponin-extracted prior to fixation. Scale bar, 20 μm. Quantitation was performed on GFP-myosin VI tail stable expressing RPE cells transfected with mock, Tom1, or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labeled with Hoechst followed by quantitation of GFP-myosin VI tail punctae/cell using an automated Cellomics VTi microscope. More than 600 cells/group from n=3 independent experiments were analysed and represented as the mean number of GFP-myosin VI tail punctae/cell (+/− s.d.).
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Figure 6: Tom1 interacts with myosin VI and is required for myosin VI localisation to endosomes(a) Myosin VI and Tom1/Tom1L2 coimmunoprecipitate from LNCAP cell lysates. Immunoprecipitation of myosin VI with three separate affinity purified rabbit polyclonal antibodies (B4, B7, 2401) was performed alongside an IgG control immunoprecipitation. The immunoprecipitates were analysed by Western blot with antibodies to Tom1/Tom1L2. (b) Binding of myosin VI to Tom1 requires the WWY motif. The mammalian two-hybrid assay was used to test binding of full-length Tom1 and Tom1L2 or various truncated versions of Tom1, annotated by amino acid numbers, against wild type myosin VI tail or the myosin VI ΔWWY or ΔRRL tail. The graph shows the mean values from n=2 independent experiments. VHS = Vps-27, Hrs and STAM, GAT = GGA and Tom1. (c) RPE cells stably expressing GFP-myosin VI were processed for immunofluorescence microscopy to evaluate GFP-myosin VI (green) and endogenous Tom1/Tom1L2 (red) or Rab5 (red) colocalisation. Inserts provide higher magnification of boxed regions. Arrows indicate areas of colocalisation. Scale bar, 20 μm. (d) RPE cells with stable expression of GFP-myosin VI tail were subjected to mock or Tom1 siRNA transfection followed by Western blot analysis and immunofluorescence microscopy to evaluate GFP-myosin VI tail localisation. Cells were either fixed directly or saponin-extracted prior to fixation. Scale bar, 20 μm. Quantitation was performed on GFP-myosin VI tail stable expressing RPE cells transfected with mock, Tom1, or TNO siRNA. Cells were processed for immunocytochemistry, immunolabelled for GFP and nuclei labeled with Hoechst followed by quantitation of GFP-myosin VI tail punctae/cell using an automated Cellomics VTi microscope. More than 600 cells/group from n=3 independent experiments were analysed and represented as the mean number of GFP-myosin VI tail punctae/cell (+/− s.d.).
Mentions: The Drosophila homologue of the Tom1 protein family, CG3529, which is an alternate endosomal sorting complex required for transport (ESCRT) class 0, was recently identified as a myosin VI binding partner23,24,25. Since the ESCRTs are required for autophagosome maturation26 , we tested whether Tom1 and myosin VI interact in mammalian cells and whether Tom1 is required for autophagy. Myosin VI and Tom1, as well as its very close homologue Tom1L2, interact in mammalian cells as they can be coimmunoprecipitated with myosin VI antibodies (Figure 6a). Using a mammalian two-hybrid assay9,10,27 we show that Tom1 binds to myosin VI via the WWY motif and not via the RRL motif, which binds optineurin, NDP52 or T6BP. The myosin VI binding site on Tom1 resides within the final 104 amino acids of the C-terminus (Figure 6b). Myosin VI and Tom1 colocalise on vesicles located along the cell periphery, that are positive for endosomal markers APPL1, GIPC, and Rab5 (Figure 6c; Supplementary Figure S6a,b). In the absence of Tom1 and Tom1L2, myosin VI is lost from endosomes (Figure 6d), and accumulates on F-actin structures throughout the cell (Supplementary Figure S7). A partial effect was observed following knockdown of the myosin VI binding partner GIPC; however, loss of T6BP, NDP52 or optineurin did not effect myosin VI localisation (Figure 6d; Supplementary Figure S6c). Thus, the Tom1 protein family is a newly identified class of myosin VI adaptor proteins that are important for targeting myosin VI to endocytic structures in mammalian cells.

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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