Hyperacetylation of α-tubulin correlates with starvation induced autophagy in tobacco BY-2 cells

Autophagy is known as an important adaptive stress response in eukaryotic cells. Also it is well known that autophagosome maturation and its intracellular transport to merge with lysosomes or lytic vacuoles are mediated by the cytoskeleton. However, the mechanism of participation of microtubules in the autophagosome formation discovered insufficiently as yet. It is assumed that regulation of cytoskeleton functional state in autophagy can be realized through posttranslational modifications of á-tubulin acetylation in particular. The objective was to study the autophagy dynamics in plant cells under metabolic stress conditions induced by long-term cultivation and functional relationship of this process with acetylation of á-tubulin. Experiments were performed using suspension culture cells of tobacco (Nicotiana tabacum L.) BY-2. Autophagy development was induced by increase in the duration of cell culture between passages (from 7 to 11 days). The first reliable signs of autophagy, namely the appearance of membrane structures stained with monodansylcadaverine were observed on 8th cultivation day and this hallmark correlated with decreasing of glucose concentration in the medium to a critical level - 1mg/ml. Following cultivation was resulted in complete glucose depletion in the medium which in turn correlated with increasing of cell number containing autophagosomes (up to 29.4% on 11th day). It should be noted that BY-2 cells demonstrate high survival rate in these stress conditions. Thus on 11th day the number of dead cells did not exceed 11% indicating the reliability of used experimental approach for simulation of autophagic processes in plant cell. Cell lysates of various cultivation stages were analyzed by Western blot using monoclonal antibodies against á-tubulin and acetylated á-tubulin. Blotted proteins were re-probed by both antibodies on the same membrane using stripping technique to quantify the acetylation level of tubulin comparing with whole tubulin amount. Following densitometric analysis revealed an essential increase of á-tubulin acetylation at the later stages of cultivation. Notably acetylation level of á-tubulin was increased in 3,5 and 11,5 folds on the 8th and 11th as compared to 7th day, respectively. Thus, glucose depletion in culture medium and the appearance of autophagosomes in BY-2 cells were correlated with á-tubulin hyperacetylation indicating the role of cytoskeleton in autophagy mediation in plant cell