Regulation of Bcl-xL activity in apoptosis

Investigation of the mechanisms in the regulation of Bcl-xL activity in apoptosis with an emphasis on the impact of posttranslational modifications on Bcl-xL stability

Bcl-xL inhibits tBid and Bax via distinct mechanisms

The proteins of the Bcl-2 family are key regulators of apoptosis. They form a complex interaction network in the cytosol and in cellular membranes, whose outcome determines mitochondrial permeabilization and commitment to death. However, we still do not understand how the action of the different family members is orchestrated to regulate apoptosis. Here, we combined quantitative analysis of the interactions and the localization dynamics of the family representatives Bcl-xL, Bax and tBid, in living cells. We discovered that Bax and tBid are able to constitutively shuttle between cytosol and mitochondria in the absence of other Bcl-2 proteins. Bcl-xL clearly stabilized tBid at mitochondria, where they formed tight complexes. In contrast, Bcl-xL promoted Bax retrotranslocation to the cytosol without affecting its shuttling rate, but by forming weak inhibitory mitochondrial complexes. Furthermore, analysis of phospho-mimetics of Bcl-xL suggested that phosphorylation regulates the function of Bcl-xL via multiple mechanisms. Altogether, our findings support a model in which the Bcl-2 network not only modulates protein/protein interactions among the family members, but also their respective intracellular localization dynamics, to regulate apoptosis

Bcl-xL inhibits tBid and Bax via distinct mechanisms

The proteins of the Bcl-2 family are key regulators of apoptosis. They form a complex interaction network in the cytosol and in cellular membranes, whose outcome determines mitochondrial permeabilization and commitment to death. However, we still do not understand how the action of the different family members is orchestrated to regulate apoptosis. Here, we combined quantitative analysis of the interactions and the localization dynamics of the family representatives Bcl-xL, Bax and tBid, in living cells. We discovered that Bax and tBid are able to constitutively shuttle between cytosol and mitochondria in the absence of other Bcl-2 proteins. Bcl-xL clearly stabilized tBid at mitochondria, where they formed tight complexes. In contrast, Bcl-xL promoted Bax retrotranslocation to the cytosol without affecting its shuttling rate, but by forming weak inhibitory mitochondrial complexes. Furthermore, analysis of phospho-mimetics of Bcl-xL suggested that phosphorylation regulates the function of Bcl-xL via multiple mechanisms. Altogether, our findings support a model in which the Bcl-2 network not only modulates protein/protein interactions among the family members, but also their respective intracellular localization dynamics, to regulate apoptosis

Early activation of CD95 is limited and localized to the cytotoxic synapse

The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti-CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction.Fil: Sánchez, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Murad, Fabronia. Universität Tübingen; AlemaniaFil: Gülcüler Balta, Gülce S.. German Cancer Research Center; Alemania. Heidelberg University; AlemaniaFil: Martin Villalba, Ana. German Cancer Research Center; AlemaniaFil: García Sáez, Ana J.. Universität Tübingen; AlemaniaFil: Carrer, Dolores Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Computed Histological Quantification of Atherosclerotic Plaque Microcalcifications

Inflammation has a central role in atherosclerotic plaque formation and rupture. Intense macrophage inflammatory activity results in microcalcifications which are strongly associated with plaque vulnerability. Microcalcifications with specific critical size between 5 and 65 mu, located in the fibrous cap producing local mechanical stress on the plaque surface and may directly contribute to plaque rupture. Hence, accurate assessment of microcalcifications size and dimension has significant clinical importance. Current invasive and noninvasive plaque imaging has limited spatial resolution which limits accurate definition of microcalcifications in the atherosclerotic plaques. We describe a new imaging technique with high spatial resolution, based on confocal microscopic analysis, using a dedicated software which allows automatic characterization of microcalcifications and quantitative assessment of their extent and localization