Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Analysis of aluminum toxicity in Hordeum vulgare roots with an emphasis on DNA integrity and cell cycle.

Barley is one of the cereals that are most sensitive to aluminum (Al). Al in acid soils limits barley growth and development and, as a result, its productivity. The inhibition of root growth is a widely accepted indicator of Al stress. Al toxicity is affected by many factors including the culture medium, pH, Al concentration and the duration of the treatment. However, Al can act differently in different species and still Al toxicity in barley deserves study. Since the mechanism of Al toxicity is discussed we cytogenetically describe the effects of different doses of bioavailable Al on the barley nuclear genome-mitotic activity, cell cycle profile and DNA integrity. At the same time, we tested an established deep-water culture (DWC) hydroponics system and analyzed the effects of Al on the root system parameters using WinRHIZO software. We demonstrated the cytotoxic and genotoxic effect of Al in barley root cells. We showed that Al treatment significantly reduced the mitotic activity of the root tip cells and it also induced micronuclei and damaged nuclei. The DNA-damaging effect of Al was observed using the TUNEL test. We define the inhibitory influence of Al on DNA replication in barley. Analysis with the labelling and detection of 5-ethynyl-2'-deoxyuridin (EdU) showed that the treatment with Al significantly decreased the frequency of S phase cells. We also demonstrated that Al exposure led to changes in the cell cycle profile of barley root tips. The delay of cell divisions observed as increased frequency of cells in G2/M phase after Al treatment was reported using flow cytometry.