Assessing mitochondrial outer membrane permeabilization during apoptosis.

Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.

Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase.

During the evolution of metazoans and the rise of systemic hormonal regulation, the insulin-controlled class 1 phosphatidylinositol 3OH-kinase (PI3K) pathway was merged with the primordial amino acid-driven mammalian target of rapamycin (mTOR) pathway to control the growth and development of the organism. Insulin regulates mTOR function through a recently described canonical signaling pathway, which is initiated by the activation of class 1 PI3K. However, how the amino acid input is integrated with that of the insulin signaling pathway is unclear. Here we used a number of molecular, biochemical, and pharmacological approaches to address this issue. Unexpectedly, we found that a major pathway by which amino acids control mTOR signaling is distinct from that of insulin and that, instead of signaling through components of the insulin/class 1 PI3K pathway, amino acids mediate mTOR activation by signaling through class 3 PI3K, hVps34.

hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase.

Mammalian cells respond to nutrient deprivation by inhibiting energy consuming processes, such as proliferation and protein synthesis, and by stimulating catabolic processes, such as autophagy. p70 S6 kinase (S6K1) plays a central role during nutritional regulation of translation. S6K1 is activated by growth factors such as insulin, and by mammalian target of rapamycin (mTOR), which is itself regulated by amino acids. The Class IA phosphatidylinositol (PI) 3-kinase plays a well recognized role in the regulation of S6K1. We now present evidence that the Class III PI 3-kinase, hVps34, also regulates S6K1, and is a critical component of the nutrient sensing apparatus. Overexpression of hVps34 or the associated hVps15 kinase activates S6K1, and insulin stimulation of S6K1 is blocked by microinjection of inhibitory anti-hVps34 antibodies, overexpression of a FYVE domain construct that sequesters the hVps34 product PI3P, or small interfering RNA-mediated knock-down of hVps34. hVps34 is not part of the insulin input to S6K1, as it is not stimulated by insulin, and inhibition of hVps34 has no effect on phosphorylation of Akt or TSC2 in insulin-stimulated cells. However, hVps34 is inhibited by amino acid or glucose starvation, suggesting that it lies on the nutrient-regulated pathway to S6K1. Consistent with this, hVps34 is also inhibited by activation of the AMP-activated kinase, which inhibits mTOR/S6K1 in glucose-starved cells. hVps34 appears to lie upstream of mTOR, as small interfering RNA knock-down of hVps34 inhibits the phosphorylation of another mTOR substrate, eIF4E-binding protein-1 (4EBP1). Our data suggest that hVps34 is a nutrient-regulated lipid kinase that integrates amino acid and glucose inputs to mTOR and S6K1.

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function.

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.