t autophagosomal bio genesis but not targeting and fusion with lysosomes. This is consistent with the report by Fass et al. However, employment of two microtubule destabilizers nocodazole and vinblastine suggest that microtubules facilitate both autophagosomal biogenesis and selleck chemicals 17-AAG fusion of autophagosomes with lysosomes. We examined whether the two drugs interfere with microtubular dynamics differently that might explain the discrepancy. Acetylated microtubules play an important role in the anterograde trafficking of vesicles. The impact of the tubulin specific histone deacetylase HDAC6 on the distribution of lysosomes suggested that microtubular acetylation may be important in autophagosome lysosome fusion.
When HeLa cells were stained with Inhibitors,Modulators,Libraries a monoclonal antibody against acetylated a tubulin that is assembled into acetylated microtubules and a polyclonal antibody against b tubulin that builds up reg ular microtubules, two sets of microtubular filaments coexisted with the acetylated microtubules that concen trated in the perinuclear region of interphase cells and on the spindles of Inhibitors,Modulators,Libraries mitotic cells. When HeLa cells were treated with increasing concentrations of dif ferent drugs, the levels of acetylated a tubulin were dra matically reduced in the presence of nocodazole, but significantly increased in the presence of vinblastine or paclitaxel. Examination of the structure of b tubulin labeled regular microtubules revealed that both nocodazole and vinblastine caused the depolymeri zation of regular microtubular filaments.
The difference was that microtubules were depolymerized into a dif fused state in the Inhibitors,Modulators,Libraries presence of nocodazole and short bar like structures in the presence of vinblastine. In contrast to microtubular depolymerization caused by nocodazole or vinblastine, paclitaxel stabilized microtubules as expected. The structures containing acetylated microtubules were affected differently by the Inhibitors,Modulators,Libraries drugs. Regu lar microtubules were depolymerised, but some fibrilar structures of acetylated microtubules remained although levels of acetylated tubulin were reduced in the presence of nocodazole. Vinblastine caused the depolymerization of not only reg ular microtubules, but also acetylated microtubules. Therefore, acetylated microtubules were nocodazole resistant but vinblastine sensitive.
Depolymerization of acetylated microtubules causes accumulation of punctate foci containing GFP LC3 Although both vinblastine and paclitaxel increased levels of acetylated a tubulin, vinblastine, but not paclitaxel caused depolymerization of acetylated micro tubules. Coincident with the breakdown of acetylated microtubules by vinblastine, the majority of vinblastine Brefeldin_A treated cells accumulated GFP www.selleckchem.com/products/epz-5676.html LC3 punctate foci that were colocalized with the dot like signals of acetylated tubulin paracrystals. Under the same condi tion, no significant more GFP punctate foci were formed upon the treatment in the autophagy defective cell line expressing GFP LC3. Less than 10% untreated or nocoda