After assembly, microtubules

After assembly, microtubules somehow are constantly modified in different patterns to enhance their functions. One type of modification is acetylation that results in acetylated microtubules that recruit molecular motors enabling increased flux of vesicles along microtubular tracks. The mammalian autophagic marker LC3 sug gests a potential role of microtubules at multiple stages in autophagy. The microtubule associated proteins MAP1A B and C19ORF5 interact with both LC3I and LC3II and facilitate their association with microtubules, suggesting an involvement of microtubules in both autophagosomal biogenesis and degradation. Previous reports suggested that microtubules are required for the trafficking of mature autophago somes.

It is still in debate whether microtu bules play a role in autophagosomal biogenesis and subsequent fusion of autophagosomes with lysosomes depends on microtubules. To decipher roles and types of microtubules in each step of autophagy, we applied a set of microtubule inter fering reagents and inhibitors of lysosomal activity to native HeLa cells or HeLa cells stably expressing the autophagic marker GFP LC3. Using both biochemical and cell biological approaches, we found that regular non acetylated microtubules are involved in autophago somal biogenesis but not required for autophagosomal degradation. It is the acetylated microtubules that are required for the fusion of autophagosomes with lyso somes to form autolysosomes.

Results Both stabilization and destabilization of microtubules impairs autophagosomal biogenesis only in mitotic cells To investigate impact of microtubules on autophagy, we created a HeLa cell line stably expressing GFP LC3 that mimics native HeLa cell line in autophagic response. As we previously reported, fewer GFP LC3 punctate rphase cells. When lysosomal activity was inhibited with NH4Cl, both interphase and mitotic cells dramatically increased numbers of punctate foci of GFP LC3 that largely colocalized with MitoTracker labeled mitochondria. Treatment with either paclitaxel or nocodazole blocked the cells in pre metaphase that carry high intensity of GFP LC3 signals. Examination of individual cells under high power microscopy revealed that more than 16% of pacli taxel treated mitotic cells contained GFP LC3 punctate foci that were colocalized with mitochondria.

This suggests that paclitaxel but not nocoda zole caused accumulation of GFP LC3 punctate foci and the accumulation only occurred in mitotic cells. The GFP LC3 pattern described Cilengitide above suggests that nocodazole increased LC3I levels while paclitaxel increased LC3II levels since the punctate foci are usually considered as the LC3II form condensed on autophago somal membranes. To confirm the idea, we separated the fraction enriched in mitotic cells by shakeoff from the attached fraction that contains both interphase and mitotic cells.

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