Lysosomal positioning coordinates cellular nutrient responses.

mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 signalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.

Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

α-Synuclein impairs macroautophagy: implications for Parkinson's disease.

Parkinson's disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication of the α-synuclein gene locus increases α-synuclein expression and causes PD. Thus, overexpression of wild-type α-synuclein is toxic. In this study, we demonstrate that α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein. Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation. Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae.

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.