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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 localises to LC3-positive autophagosomes(a) RPE cells transiently cotransfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 hours (bottom row) were processed for immunofluorescence microscopy. Arrows highlight areas of colocalisation and inserts provide higher magnification of boxed regions. Scale bar, 20 μm. (b) RPE cells treated with 100 nM BafilomycinA1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 colocalisation. Arrows highlight areas of colocalisation. Scale bar, 20 μm. (c) Image stills acquired from spinning disk time-lapse video microscopy of RPE cells transiently cotransfected with cherry-LC3 and GFP-myosin VI. Boxed regions highlight areas of interest within the cell that are shown to the right (i, ii, iii) as video image stills captured at 1-minute intervals. Arrowheads highlight myosin VI positive vesicles (green) coming into contact and colocalising with LC3-positive vesicles (red). Scale bar, 10 μm (whole cell image), 2 μm (cropped images). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualised with phalloidin (white). Nuclei were labelled with Hoechst (blue). Arrowheads indicate areas of actin rich myosin VI/LC3-positive autophagosomes. Scale bar, 20 μm
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Figure 3: Myosin VI localises to LC3-positive autophagosomes(a) RPE cells transiently cotransfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 hours (bottom row) were processed for immunofluorescence microscopy. Arrows highlight areas of colocalisation and inserts provide higher magnification of boxed regions. Scale bar, 20 μm. (b) RPE cells treated with 100 nM BafilomycinA1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 colocalisation. Arrows highlight areas of colocalisation. Scale bar, 20 μm. (c) Image stills acquired from spinning disk time-lapse video microscopy of RPE cells transiently cotransfected with cherry-LC3 and GFP-myosin VI. Boxed regions highlight areas of interest within the cell that are shown to the right (i, ii, iii) as video image stills captured at 1-minute intervals. Arrowheads highlight myosin VI positive vesicles (green) coming into contact and colocalising with LC3-positive vesicles (red). Scale bar, 10 μm (whole cell image), 2 μm (cropped images). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualised with phalloidin (white). Nuclei were labelled with Hoechst (blue). Arrowheads indicate areas of actin rich myosin VI/LC3-positive autophagosomes. Scale bar, 20 μm

Mentions: To further characterise the precise role of this motor protein in the autophagocytic pathway, we next analysed myosin VI targeting and localisation to autophagosomes. In human RPE cells, GFP-myosin VI is recruited to cherry-LC3-positive autophagosomes (Figure 3a) and endogenous myosin VI colocalises with endogenous LC3 (Figure 3b) under conditions that induce nonselective autophagy such as treatment with Torin1, a selective mTOR kinase inhibitor, or following amino acid withdrawal (Supplementary Figure S4a). Furthermore, myosin VI colocalises with LC3-positive autophagosomes containing the aggregate prone protein Ataxin3 with an 84 amino acid polyglutamine repeat, Ataxin3Q84 (Supplementary Figure S4b), highlighting a role for myosin VI in nonselective as well as in selective autophagy. A high proportion of LC3-positive autophagosomes colocalise with myosin VI (LC3/MVI), but only a distinct subpopulation of myosin VI is present on LC3-positive vesicles (MVI/LC3), reflecting the association of this multifunctional motor not only with autophagosomes but also with endocytic and secretory membrane trafficking compartments (Supplementary Figure S4c). Often, the localisation of myosin VI does not completely overlap with that of LC3 on autophagosomes, but is present in distinct areas or subdomains of the autophagosomal membrane (Figure 3a,b), reminiscent of a small membrane vesicle docking at a larger autophagocytic vacuole.


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 localises to LC3-positive autophagosomes(a) RPE cells transiently cotransfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 hours (bottom row) were processed for immunofluorescence microscopy. Arrows highlight areas of colocalisation and inserts provide higher magnification of boxed regions. Scale bar, 20 μm. (b) RPE cells treated with 100 nM BafilomycinA1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 colocalisation. Arrows highlight areas of colocalisation. Scale bar, 20 μm. (c) Image stills acquired from spinning disk time-lapse video microscopy of RPE cells transiently cotransfected with cherry-LC3 and GFP-myosin VI. Boxed regions highlight areas of interest within the cell that are shown to the right (i, ii, iii) as video image stills captured at 1-minute intervals. Arrowheads highlight myosin VI positive vesicles (green) coming into contact and colocalising with LC3-positive vesicles (red). Scale bar, 10 μm (whole cell image), 2 μm (cropped images). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualised with phalloidin (white). Nuclei were labelled with Hoechst (blue). Arrowheads indicate areas of actin rich myosin VI/LC3-positive autophagosomes. Scale bar, 20 μm
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Figure 3: Myosin VI localises to LC3-positive autophagosomes(a) RPE cells transiently cotransfected with GFP-myosin VI and cherry-LC3 and either left untreated (top row) or treated with 250 nM Torin1 for 2 hours (bottom row) were processed for immunofluorescence microscopy. Arrows highlight areas of colocalisation and inserts provide higher magnification of boxed regions. Scale bar, 20 μm. (b) RPE cells treated with 100 nM BafilomycinA1 were processed for immunofluorescence microscopy to evaluate endogenous myosin VI and endogenous LC3 colocalisation. Arrows highlight areas of colocalisation. Scale bar, 20 μm. (c) Image stills acquired from spinning disk time-lapse video microscopy of RPE cells transiently cotransfected with cherry-LC3 and GFP-myosin VI. Boxed regions highlight areas of interest within the cell that are shown to the right (i, ii, iii) as video image stills captured at 1-minute intervals. Arrowheads highlight myosin VI positive vesicles (green) coming into contact and colocalising with LC3-positive vesicles (red). Scale bar, 10 μm (whole cell image), 2 μm (cropped images). (d) Confocal immunofluorescence microscopy of RPE cells stably expressing cherry-LC3 following transient transfection with GFP-myosin VI. Immunostaining for GFP (green) and cherry (red) was performed and actin was visualised with phalloidin (white). Nuclei were labelled with Hoechst (blue). Arrowheads indicate areas of actin rich myosin VI/LC3-positive autophagosomes. Scale bar, 20 μm
Mentions: To further characterise the precise role of this motor protein in the autophagocytic pathway, we next analysed myosin VI targeting and localisation to autophagosomes. In human RPE cells, GFP-myosin VI is recruited to cherry-LC3-positive autophagosomes (Figure 3a) and endogenous myosin VI colocalises with endogenous LC3 (Figure 3b) under conditions that induce nonselective autophagy such as treatment with Torin1, a selective mTOR kinase inhibitor, or following amino acid withdrawal (Supplementary Figure S4a). Furthermore, myosin VI colocalises with LC3-positive autophagosomes containing the aggregate prone protein Ataxin3 with an 84 amino acid polyglutamine repeat, Ataxin3Q84 (Supplementary Figure S4b), highlighting a role for myosin VI in nonselective as well as in selective autophagy. A high proportion of LC3-positive autophagosomes colocalise with myosin VI (LC3/MVI), but only a distinct subpopulation of myosin VI is present on LC3-positive vesicles (MVI/LC3), reflecting the association of this multifunctional motor not only with autophagosomes but also with endocytic and secretory membrane trafficking compartments (Supplementary Figure S4c). Often, the localisation of myosin VI does not completely overlap with that of LC3 on autophagosomes, but is present in distinct areas or subdomains of the autophagosomal membrane (Figure 3a,b), reminiscent of a small membrane vesicle docking at a larger autophagocytic vacuole.

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