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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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Loss of myosin VI function leads to an accumulation of autophagosomes(a) Western blot analysis of myosin VI depleted Hela cells untreated or treated with 100 nM Bafilomycin A1. (b) Quantitation of Western blot LC3-II intensity (+/− s.d.) (n=3). **p<0.01, ***p<0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in Hela cells following knockdown of myosin VI. Hoechst labels nuclei (blue). Scale bar, 20 μm. (d) Quantitation of LC3- and p62-positive punctae was performed and results are represented as average punctae fluorescence intensity/cell (+/− s.d.) (n=3) from >1500 cells/experiment. (e) Western blot analysis of parental Hela cells or Hela cells stably expressing siRNA resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of mouse embryonic fibroblasts cultured from wild-type and Snell’s Waltzer (SV) mice treated with 1 μM MG132 or 100 nM Bafilomycin A1. (g) Quantitation of Western blot LC3-II intensity (+/− s.d) (n=3). **p<0.01. (h) To evaluate effects on autophagosome biogenesis, results are displayed as the fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (+/− s.d.) (n=3) (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM BafilomycinA1. (j) Quantitation of Western blot LC3-II intensity was performed. (n=2) (k) To evaluate effects on autophagosome biogenesis, results are displayed as fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (n=2) (l) Confocal immunofluorescence microscopy of mock or myosin VI siRNA treated Hela cells stably expressing RFP-GFP-LC3 reporter. Hoechst labels nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock or myosin VI siRNA treated Hela cells expressing RFP-GFP-LC3. Data is represented as the Pearson’s coefficient of RFP and GFP signal correlation from >100 cells/experiment. (+/− s.d.) (n=3) Scale bar, 20 μm. (n) Confocal immunofluorescence microscopy of myosin VI depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm.
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Figure 1: Loss of myosin VI function leads to an accumulation of autophagosomes(a) Western blot analysis of myosin VI depleted Hela cells untreated or treated with 100 nM Bafilomycin A1. (b) Quantitation of Western blot LC3-II intensity (+/− s.d.) (n=3). **p<0.01, ***p<0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in Hela cells following knockdown of myosin VI. Hoechst labels nuclei (blue). Scale bar, 20 μm. (d) Quantitation of LC3- and p62-positive punctae was performed and results are represented as average punctae fluorescence intensity/cell (+/− s.d.) (n=3) from >1500 cells/experiment. (e) Western blot analysis of parental Hela cells or Hela cells stably expressing siRNA resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of mouse embryonic fibroblasts cultured from wild-type and Snell’s Waltzer (SV) mice treated with 1 μM MG132 or 100 nM Bafilomycin A1. (g) Quantitation of Western blot LC3-II intensity (+/− s.d) (n=3). **p<0.01. (h) To evaluate effects on autophagosome biogenesis, results are displayed as the fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (+/− s.d.) (n=3) (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM BafilomycinA1. (j) Quantitation of Western blot LC3-II intensity was performed. (n=2) (k) To evaluate effects on autophagosome biogenesis, results are displayed as fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (n=2) (l) Confocal immunofluorescence microscopy of mock or myosin VI siRNA treated Hela cells stably expressing RFP-GFP-LC3 reporter. Hoechst labels nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock or myosin VI siRNA treated Hela cells expressing RFP-GFP-LC3. Data is represented as the Pearson’s coefficient of RFP and GFP signal correlation from >100 cells/experiment. (+/− s.d.) (n=3) Scale bar, 20 μm. (n) Confocal immunofluorescence microscopy of myosin VI depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm.

Mentions: To understand the role of myosin VI and its binding partners in autophagy, we first assessed whether loss of myosin VI, in the myosin VI knockout mouse or by siRNA-mediated knockdown (KD), affects cellular autophagy levels by measuring the abundance of the lipidated autophagosome-associated LC3 (LC3-II)16. In Hela cells, myosin VI expression was suppressed with siRNA and conversion of LC3-I to LC3-II quantified in the presence or absence of the vacuolar-type H+-ATPase inhibitor BafilomycinA1, which blocks fusion of autophagosomes with lysosomes. There is a significant increase in the amount of LC3-II in myosin VI KD cells under steady state conditions as well as in response to autophagy induction following treatment with the proteasome inhibitor MG132 (Figure 1a,b; Supplementary Figure S1a). Elevated levels of LC3-II indicate an accumulation of autophagosomes, which was not due to an increase in autophagosome biogenesis, since there was no increase in LC3-II in the presence of Bafilomycin A1 (Figure 1a,b). We also observed by immunofluorescence microscopy an accumulation of LC3-positive autophagosomes and the autophagy cargo receptor p62 under basal conditions in myosin VI knockdown cells (Figure 1c,d; Supplementary Figure S1b). These results were verified using single siRNA oligonucleotides targeting myosin VI (Supplementary Figure S1c) and by rescue experiments using a Hela cell line stably expressing GFP-myosin VI containing silent mutations in the target region of a single siRNA oligonucleotide (Figure 1e). Loss of myosin VI expression in parental cells leads to an accumulation of LC3-II (Figure 1a,e), while in rescue cells expressing siRNA resistant myosin VI, loss of endogenous myosin VI has no effect on LC3-II levels (Figure 1e). Interestingly, loss of myosin VI expression also leads to enlarged, swollen GFP-LC3-positive autophagosomes (Supplementary Figure S1d,S2a), which increased from an average area of 1.39+/−0.049 (s.d.) μm2 in mock treated cells to 1.57+/− 0.055 (s.d) μm2 in myosin VI depleted cells (Supplementary Figure S1e).


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)

Loss of myosin VI function leads to an accumulation of autophagosomes(a) Western blot analysis of myosin VI depleted Hela cells untreated or treated with 100 nM Bafilomycin A1. (b) Quantitation of Western blot LC3-II intensity (+/− s.d.) (n=3). **p<0.01, ***p<0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in Hela cells following knockdown of myosin VI. Hoechst labels nuclei (blue). Scale bar, 20 μm. (d) Quantitation of LC3- and p62-positive punctae was performed and results are represented as average punctae fluorescence intensity/cell (+/− s.d.) (n=3) from >1500 cells/experiment. (e) Western blot analysis of parental Hela cells or Hela cells stably expressing siRNA resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of mouse embryonic fibroblasts cultured from wild-type and Snell’s Waltzer (SV) mice treated with 1 μM MG132 or 100 nM Bafilomycin A1. (g) Quantitation of Western blot LC3-II intensity (+/− s.d) (n=3). **p<0.01. (h) To evaluate effects on autophagosome biogenesis, results are displayed as the fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (+/− s.d.) (n=3) (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM BafilomycinA1. (j) Quantitation of Western blot LC3-II intensity was performed. (n=2) (k) To evaluate effects on autophagosome biogenesis, results are displayed as fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (n=2) (l) Confocal immunofluorescence microscopy of mock or myosin VI siRNA treated Hela cells stably expressing RFP-GFP-LC3 reporter. Hoechst labels nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock or myosin VI siRNA treated Hela cells expressing RFP-GFP-LC3. Data is represented as the Pearson’s coefficient of RFP and GFP signal correlation from >100 cells/experiment. (+/− s.d.) (n=3) Scale bar, 20 μm. (n) Confocal immunofluorescence microscopy of myosin VI depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm.
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Figure 1: Loss of myosin VI function leads to an accumulation of autophagosomes(a) Western blot analysis of myosin VI depleted Hela cells untreated or treated with 100 nM Bafilomycin A1. (b) Quantitation of Western blot LC3-II intensity (+/− s.d.) (n=3). **p<0.01, ***p<0.001 (c) Confocal immunofluorescence microscopy of LC3 punctae (green) in Hela cells following knockdown of myosin VI. Hoechst labels nuclei (blue). Scale bar, 20 μm. (d) Quantitation of LC3- and p62-positive punctae was performed and results are represented as average punctae fluorescence intensity/cell (+/− s.d.) (n=3) from >1500 cells/experiment. (e) Western blot analysis of parental Hela cells or Hela cells stably expressing siRNA resistant GFP-myosin VI transiently transfected with a single myosin VI siRNA oligonucleotide following treatment with 1 μM MG132. (f) Western blot analysis of mouse embryonic fibroblasts cultured from wild-type and Snell’s Waltzer (SV) mice treated with 1 μM MG132 or 100 nM Bafilomycin A1. (g) Quantitation of Western blot LC3-II intensity (+/− s.d) (n=3). **p<0.01. (h) To evaluate effects on autophagosome biogenesis, results are displayed as the fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (+/− s.d.) (n=3) (i) Western blot analysis of cortical neurons from wild-type and SV mice treated with 1 μM MG132 in the absence or presence of 100 nM BafilomycinA1. (j) Quantitation of Western blot LC3-II intensity was performed. (n=2) (k) To evaluate effects on autophagosome biogenesis, results are displayed as fold increase in normalised LC3-II intensity with Bafilomycin A1 compared to untreated control. (n=2) (l) Confocal immunofluorescence microscopy of mock or myosin VI siRNA treated Hela cells stably expressing RFP-GFP-LC3 reporter. Hoechst labels nuclei (blue). (m) Quantitative data of RFP and GFP signal overlap from confocal images of mock or myosin VI siRNA treated Hela cells expressing RFP-GFP-LC3. Data is represented as the Pearson’s coefficient of RFP and GFP signal correlation from >100 cells/experiment. (+/− s.d.) (n=3) Scale bar, 20 μm. (n) Confocal immunofluorescence microscopy of myosin VI depleted RPE cells stably expressing GFP-LC3 immunostained against GFP (green) and Cathepsin D (red). Hoechst labels nuclei (blue). Insets represent magnified boxed regions. Scale bar, 20 μm.
Mentions: To understand the role of myosin VI and its binding partners in autophagy, we first assessed whether loss of myosin VI, in the myosin VI knockout mouse or by siRNA-mediated knockdown (KD), affects cellular autophagy levels by measuring the abundance of the lipidated autophagosome-associated LC3 (LC3-II)16. In Hela cells, myosin VI expression was suppressed with siRNA and conversion of LC3-I to LC3-II quantified in the presence or absence of the vacuolar-type H+-ATPase inhibitor BafilomycinA1, which blocks fusion of autophagosomes with lysosomes. There is a significant increase in the amount of LC3-II in myosin VI KD cells under steady state conditions as well as in response to autophagy induction following treatment with the proteasome inhibitor MG132 (Figure 1a,b; Supplementary Figure S1a). Elevated levels of LC3-II indicate an accumulation of autophagosomes, which was not due to an increase in autophagosome biogenesis, since there was no increase in LC3-II in the presence of Bafilomycin A1 (Figure 1a,b). We also observed by immunofluorescence microscopy an accumulation of LC3-positive autophagosomes and the autophagy cargo receptor p62 under basal conditions in myosin VI knockdown cells (Figure 1c,d; Supplementary Figure S1b). These results were verified using single siRNA oligonucleotides targeting myosin VI (Supplementary Figure S1c) and by rescue experiments using a Hela cell line stably expressing GFP-myosin VI containing silent mutations in the target region of a single siRNA oligonucleotide (Figure 1e). Loss of myosin VI expression in parental cells leads to an accumulation of LC3-II (Figure 1a,e), while in rescue cells expressing siRNA resistant myosin VI, loss of endogenous myosin VI has no effect on LC3-II levels (Figure 1e). Interestingly, loss of myosin VI expression also leads to enlarged, swollen GFP-LC3-positive autophagosomes (Supplementary Figure S1d,S2a), which increased from an average area of 1.39+/−0.049 (s.d.) μm2 in mock treated cells to 1.57+/− 0.055 (s.d) μm2 in myosin VI depleted cells (Supplementary Figure S1e).

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