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Acetylated microtubules are required for fusion of autophagosomes with lysosomes

Xie, Rui, Nguyen, Susan, McKeehan, Wallace L, Liu, Leyuan·2010·BMC Cell Biology
DOI 10.1186/1471-2121-11-89PMC PMC2995476
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Abstract

Background: Autophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. Although it is agreed that the LC3II-associated mature autophagosomes move along microtubular tracks, it is still in dispute if the conversion of LC3I to LC3II before autophagosomes are fully mature and subsequent fusion of mature autophagosomes with lysosomes require microtubules. Results: We use biochemical markers of autophagy and a collection of microtubule interfering reagents to test the question. Results show that interruption of microtubules with either microtubule stabilizing paclitaxel or destabilizing nocodazole similarly impairs the conversion of LC3I to LC3II, but does not block the degradation of LC3II-associated autophagosomes. Acetylation of microtubules renders them resistant to nocodazole treatment. Treatment with vinblastine that causes depolymerization of both non-acetylated and acetylated microtubules results in impairment of both LC3I-LC3II conversion and LC3II-associated autophagosome fusion with lysosomes. Conclusions: Acetylated microtubules are required for fusion of autophagosomes with lysosomes to form autolysosomes.

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Figure 1 Only paclitaxel causes accumulation of punctate foci of GFP-LC3 in mitotic cells. (A) Accumulation of GFP-LC3 punctate foci in cells treated with either paclitaxel or nocodazole overnight. Mitochondria are shown in red after exposure to MitoTracker Red CMXRos dye [32,39]. The punctate foci of bright green GFP-LC3 in stably transfected HeLa cells overlapping with weak red punctate foci of dysfunctional mitochondria are indicative of mitophagosomes or mitolysosomes (mitochondria-containing autolysosomes). Colocalized punctate foci in white were revealed by analysis with an ImageJ ColocalizeRGB Plugin. The GFP-LC3 punctate foci overlapping with the much weaker signal of MitoTracker were not detected in the colocalization analysis. Lysosomal inhibitor NH4Cl (20 mM NH4Cl) was added simultaneously to preserve the resulting mitophagosomes. Scale bar, 10 μm in all panels. Ctrl, Control, Noc, 10 μM nocodazole; Tax, 10 μM paclitaxel. (B) Fluorescent intensity of GFP-LC3 in mitotic cells treated with paclitaxel and nocodazole overnight. (C) The percentage of pro-metaphase cells with punctate foci of GFP-LC3 as shown for the mitotic cells treated with paclitaxel overnight in the absence of NH4Cl (A). Number of cells with GFP-LC3 punctate foci was counted directly and expressed as a percentage of total mitotic cells. Because of the difference in frequency of mitosis, about 100 mitotic cells in 20 to 50 different fields were scored in the presence or absence of nocodazole or paclitaxel, respectively. The data represent the means and standard errors of three independent experiments. **, p < 0.01 as calculated by Student's T-test.
Figure 1 Only paclitaxel causes accumulation of punctate foci of GFP-LC3 in mitotic cells. (A) Accumulation of GFP-LC3 punctate foci in cells treated with either paclitaxel or nocodazole overnight. Mitochondria are shown in red after exposure to MitoTracker Red CMXRos dye [32,39]. The punctate foci of bright green GFP-LC3 in stably transfected HeLa cells overlapping with weak red punctate foci of dysfunctional mitochondria are indicative of mitophagosomes or mitolysosomes (mitochondria-containing autolysosomes). Colocalized punctate foci in white were revealed by analysis with an ImageJ ColocalizeRGB Plugin. The GFP-LC3 punctate foci overlapping with the much weaker signal of MitoTracker were not detected in the colocalization analysis. Lysosomal inhibitor NH4Cl (20 mM NH4Cl) was added simultaneously to preserve the resulting mitophagosomes. Scale bar, 10 μm in all panels. Ctrl, Control, Noc, 10 μM nocodazole; Tax, 10 μM paclitaxel. (B) Fluorescent intensity of GFP-LC3 in mitotic cells treated with paclitaxel and nocodazole overnight. (C) The percentage of pro-metaphase cells with punctate foci of GFP-LC3 as shown for the mitotic cells treated with paclitaxel overnight in the absence of NH4Cl (A). Number of cells with GFP-LC3 punctate foci was counted directly and expressed as a percentage of total mitotic cells. Because of the difference in frequency of mitosis, about 100 mitotic cells in 20 to 50 different fields were scored in the presence or absence of nocodazole or paclitaxel, respectively. The data represent the means and standard errors of three independent experiments. **, p < 0.01 as calculated by Student's T-test.CC BY · PubMed Central
Figure 2 Interrupting the dynamics of regular microtubules with either nocodazole or paclitaxel impairs conversion of LC3I to LC3II only in mitotic cells. (A) Immunoblot of lysates of fractioned HeLa cells. Mitotic cells were enriched by synchronization with 2 mM thymidine for 20 hr followed by release from the thymidine blockade for 20 hr in the absence or presence of 10 μM paclitaxel or nocodazole as described previously [40]. NH4Cl (20 mM) was added simultaneously with microtubule interfering agents. Loosely attached mitotic cells (S) were harvested by vigorous shake off from the remaining mixture (A) of interphase cells and mitotic cells attached to the dish. (B) The relative intensities of LC3I and LC3II bands were calculated from the scanned intensities of bands of three independent experiments as exemplified by immunoblot shown in (A). **, p < 0.01 as calculated by Student's T-test.
Figure 2 Interrupting the dynamics of regular microtubules with either nocodazole or paclitaxel impairs conversion of LC3I to LC3II only in mitotic cells. (A) Immunoblot of lysates of fractioned HeLa cells. Mitotic cells were enriched by synchronization with 2 mM thymidine for 20 hr followed by release from the thymidine blockade for 20 hr in the absence or presence of 10 μM paclitaxel or nocodazole as described previously [40]. NH4Cl (20 mM) was added simultaneously with microtubule interfering agents. Loosely attached mitotic cells (S) were harvested by vigorous shake off from the remaining mixture (A) of interphase cells and mitotic cells attached to the dish. (B) The relative intensities of LC3I and LC3II bands were calculated from the scanned intensities of bands of three independent experiments as exemplified by immunoblot shown in (A). **, p < 0.01 as calculated by Student's T-test.CC BY · PubMed Central
Figure 3 Impact of paclitaxel, vinblastine and nocodazole on microtubular acetylation and structure. (A) Immunostaining of microtubules in HeLa cells untreated or treated with 10 μM of nocodazole, vinblastine or paclitaxel overnight with antibodies against acetylated α-tubulin and normal β-tubulin. Ac-Tub, acetylated α-tubulin. β-Tub, β-Tubulin. Vin, vinblastine. (B) Immunoblotting analysis of acetylated α-tubulin levels in lysates of HeLa cells treated with different concentrations of nocodazole, vinblastine and paclitaxel for overnight.
Figure 3 Impact of paclitaxel, vinblastine and nocodazole on microtubular acetylation and structure. (A) Immunostaining of microtubules in HeLa cells untreated or treated with 10 μM of nocodazole, vinblastine or paclitaxel overnight with antibodies against acetylated α-tubulin and normal β-tubulin. Ac-Tub, acetylated α-tubulin. β-Tub, β-Tubulin. Vin, vinblastine. (B) Immunoblotting analysis of acetylated α-tubulin levels in lysates of HeLa cells treated with different concentrations of nocodazole, vinblastine and paclitaxel for overnight.CC BY · PubMed Central
Figure 4 Vinblastine-induced depolymerization of acetylated microtubules is coincident with accumulation of GFP-LC3 punctate foci. (A, B) Accumulation of GFP punctate foci in HeLa cells stably expressing GFP-LC3 (A) or GFP-ΔLC3 (B) untreated or treated with either nocodazole or vinblastine in the absence or presence of lysosomal inhibitor bafilomycin A1 (BAF) overnight. The acetylated microtubules are counterstained with anti-acetylated α-tubulin antibody. (C) Percentage of GFP expressing cells with more than 10 GFP punctate foci. Only GFP punctate foci occupying an area with diameter greater than four pixels (~0.15 μm) were counted. The data is the average and standard deviation of three independent experiments. **, p < 0.01 as calculated by Student's T-test.
Figure 4 Vinblastine-induced depolymerization of acetylated microtubules is coincident with accumulation of GFP-LC3 punctate foci. (A, B) Accumulation of GFP punctate foci in HeLa cells stably expressing GFP-LC3 (A) or GFP-ΔLC3 (B) untreated or treated with either nocodazole or vinblastine in the absence or presence of lysosomal inhibitor bafilomycin A1 (BAF) overnight. The acetylated microtubules are counterstained with anti-acetylated α-tubulin antibody. (C) Percentage of GFP expressing cells with more than 10 GFP punctate foci. Only GFP punctate foci occupying an area with diameter greater than four pixels (~0.15 μm) were counted. The data is the average and standard deviation of three independent experiments. **, p < 0.01 as calculated by Student's T-test.CC BY · PubMed Central
Figure 5 Only vinblastine causes a blockade of LC3II and P62 degradation. (A) A representative immunoblot of lysates of HeLa cells treated with different concentration of drugs in the absence or presence of lysosomal inhibitor bafilomycin A1 overnight. P62, P62 (SQSTM1). (B) Plots of the relative intensity of LC3II and P62 bands detected from three independent blots as represented by the one shown in (A). **, p < 0.01 as calculated by Student's T-test.
Figure 5 Only vinblastine causes a blockade of LC3II and P62 degradation. (A) A representative immunoblot of lysates of HeLa cells treated with different concentration of drugs in the absence or presence of lysosomal inhibitor bafilomycin A1 overnight. P62, P62 (SQSTM1). (B) Plots of the relative intensity of LC3II and P62 bands detected from three independent blots as represented by the one shown in (A). **, p < 0.01 as calculated by Student's T-test.CC BY · PubMed Central
Figure 6 Vinblastine blocks autophagosome-lysosome fusion on acetylated microtubules. (A) Immunoblotting analysis of LAMP2 levels in HeLa cells treated with different concentrations of nocodazole and vinblastine in the absence or presence of bafilomycin A1 overnight. (B) Colocalization analysis of GFP-LC3 punctate foci with lysosomal marker LAMP2 in HeLa cells stably expressing GFP-LC3 treated with nocodazole or vinblastine in the presence of bafilomycin A1 overnight. LAMP2 was visualized with anti-LAMP2 primary antibody and Rhodamine-conjugated secondary antibody. The overlapping analysis was performed with the ImageJ ColocalizeRGB Plugins. (C) The fraction of GFP-LC3 overlapping with LAMP2 staining as calculated from images shown in (B) with the ImageJ JACoP Plugins. The data shown is the average and standard deviation of ten randomly selected images in a field of 512 pixels × 512 pixels (23.5 μm × 23.5 μm). The significance of differences was determined by Student's t-test.
Figure 6 Vinblastine blocks autophagosome-lysosome fusion on acetylated microtubules. (A) Immunoblotting analysis of LAMP2 levels in HeLa cells treated with different concentrations of nocodazole and vinblastine in the absence or presence of bafilomycin A1 overnight. (B) Colocalization analysis of GFP-LC3 punctate foci with lysosomal marker LAMP2 in HeLa cells stably expressing GFP-LC3 treated with nocodazole or vinblastine in the presence of bafilomycin A1 overnight. LAMP2 was visualized with anti-LAMP2 primary antibody and Rhodamine-conjugated secondary antibody. The overlapping analysis was performed with the ImageJ ColocalizeRGB Plugins. (C) The fraction of GFP-LC3 overlapping with LAMP2 staining as calculated from images shown in (B) with the ImageJ JACoP Plugins. The data shown is the average and standard deviation of ten randomly selected images in a field of 512 pixels × 512 pixels (23.5 μm × 23.5 μm). The significance of differences was determined by Student's t-test.CC BY · PubMed Central

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Metadata

Authors
Xie, R., Nguyen, S., McKeehan, W. L., & Liu, L.
Venue
BMC Cell Biology
Publisher
Springer Science and Business Media LLC
Published
2010-11-22
Volume
11
Issue
1
ISSN
1471-2121
DOI
10.1186/1471-2121-11-89
PMC
PMC2995476
Cited by
195
References
11
Language
en

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