A variety of physiological death signals, as well as pathological cellular insults, trigger the genetically programmed pathway of apoptosis (Vaux and Korsmeyer 1999). Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction, of which mitochondrial dysfunction is the best characterized (for reviews, see Green and Reed 1998; Thornberry and Lazebnik 1998). As the BCL-2 family members reside upstream of irreversible cellular damage and focus much of their efforts at the level of mitochondria, they play a pivotal role in deciding whether a cell will live or die (Fig. 1). The founder of this family, the BCL-2 proto-oncogene, was discovered at the chromosomal breakpoint of t(14;18) bearing human B-cell lymphomas. The BCL-2 family of proteins has expanded significantly and includes both proas well as anti-apoptotic molecules. Indeed, the ratio between these two subsets helps determine, in part, the susceptibility of cells to a death signal (Oltvai et al. 1993) (Fig. 1). An additional characteristic of the members of this family is their frequent ability to form homoas well as heterodimers, suggesting neutralizing competition between these proteins. A further characteristic of probable functional significance is their ability to become integral membrane proteins. BCL-2 family members possess up to four conserved BCL-2 homology (BH) domains designated BH1, BH2, BH3, and BH4, which correspond to a-helical segments (Adams and Cory 1998; Kelekar and Thompson 1998; Reed 1998) (Fig. 2). Many of the anti-apoptotic members display sequence conservation in all four domains. The pro-apoptotic molecules frequently display less sequence conservation of the first a-helical segment, BH4. Deletion and mutagenesis studies argue that the amphipathic a-helical BH3 domain serves as a critical death domain in the pro-apoptotic members. This concept is supported by an emerging subset of “BH3-domain-only” members who display sequence homology only within the BH3 domain and to date are all pro-apoptotic. However, the three-dimensional structure of at least one BH3-domainonly molecule, BID, demonstrates a very similar overall a-helical content to the anti-apoptotic molecule BCL-XL (Chou et al. 1999; McDonnell et al. 1999). Many BCL-2 family members also contain a carboxy-terminal hydrophobic domain, which in the case of BCL-2 is essential for its targeting to membranes such as the mitochondrial outer membrane (Nguyen et al. 1993).

/pdf/bcl-2-family-members-and-the-mitochondria-in-apoptosis-45he6ri17y.pdf

BCL-2 family members and the mitochondria in apoptosis

Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.

/pdf/studying-protein-dynamics-in-living-cells-2jib9fauyq.pdf

Studying protein dynamics in living cells.

Mitochondria play a central role in the survival and death of neurons. The detailed bioenergetic mechanisms by which isolated mitochondria generate ATP, sequester Ca2+, generate reactive oxygen species, and undergo Ca2+-dependent permeabilization of their inner membrane are currently being applied to the function of mitochondria in situ within neurons under physiological and pathophysiological conditions. Here we review the functional bioenergetics of isolated mitochondria, with emphasis on the chemiosmotic proton circuit and the application (and occasional misapplication) of these principles to intact neurons. Mitochondria play an integral role in both necrotic and apoptotic neuronal cell death, and the bioenergetic principles underlying current studies are reviewed.

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.1.315

Mitochondria and Neuronal Survival

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants

The Bcl-2 protein family: sensors and checkpoints for life-or-death decisions.

It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.

Roger Heim

Papers

Bcl-2 and bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer