Microtubules are a key component of autophagy and health, and only dry fasting fixes them

Autophagosome completion and fusion

In what is perhaps the least understood step of macroautophagy, the expanding phagophore must eventually mature and close to form a completed autophagosome, which traffics to and fuses with an endosome and/or lysosome, becoming an autolysosome. Movement of autophagosomes to lysosomes is dependent on microtubules (108). Fusion of autophagosomes with endosomes involves the protein VTIlB (5). UVRAG, which can associate with the PtdIns3K complex, can activate the GTPase RAB7, which promotes fusion with lysosomes (59, 86). It has also been suggested that components of the SNARE machinery, such as VAM7 and VAM9, have a role in fusion (28, 31). Recent work has identified another SNARE, syntaxin 17, which localizes to completed autophagosomes and is required for fusion with the endosome/lysosome through an interaction with SNAP29 and the endosomal/lysosomal SNARE VAMP8 (56).

Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.

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