Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor.

The interface between the endoplasmic reticulum (ER) and mitochondria referred to as the MAM (mitochondria-associated ER membrane) plays important roles in many physiological functions. A specific marker for this important entity of cellular structure is urgently needed. Thus, we propose in this method chapter that the membrane-bound ER chaperone sigma-1 receptor serves as an ideal marker for the MAM. We describe in detail the preparation and purification of the MAM by using the sigma-1 receptor as the marker and demonstrate the uniqueness of this marker by using a variety of cells, peripheral and neuronal.

The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism.

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. (13)C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive (13)C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.

Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival.

Bcl-2 family proteins, known for their apoptosis functioning at the mitochondria, have been shown to localize to other cellular compartments to mediate calcium (Ca2+) signals. Since the proper supply of Ca2+ in cells serves as an important mechanism for cellular survival and bioenergetics, we propose an integrating role for Bcl-2 family proteins in modulating Ca2+ signaling. The endoplasmic reticulum (ER) is the main Ca2+ storage for the cell and Bcl-2 family proteins competitively regulate its Ca2+ concentration. Bcl-2 family proteins also regulate the flux of Ca2+ from the ER by physically interacting with inositol 1,4,5-trisphosphate receptors (IP3Rs) to mediate their opening. Type 1 IP3Rs reside at the bulk ER to coordinate cytosolic Ca2+ signals, while type 3 IP3Rs reside at mitochondria-associated ER membrane (MAM) to facilitate mitochondrial Ca2+ uptake. In healthy cells, mitochondrial Ca2+ drives pyruvate into the citric acid (TCA) cycle to facilitate ATP production, while a continuous accumulation of Ca2+ can trigger the release of cytochrome c, thus initiating apoptosis. Since multiple organelles and Bcl-2 family proteins are involved in Ca2+ signaling, we aim to clarify the role that Bcl-2 family proteins play in facilitating Ca2+ signaling and how mitochondrial Ca2+ is relevant in both bioenergetics and apoptosis. We also explore how these insights could be useful in controlling bioenergetics in apoptosis-resistant cell lines.

Antigen retrieval to improve the immunocytochemistry detection of sigma-1 receptors and ER chaperones.

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80°C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 h effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures.