The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes.

Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins.

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7 cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7 cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

Efficacy and specificity of inhibitors of BCL-2 family protein interactions assessed by affinity measurements in live cells.

Cytoplasmic and membrane-bound BCL-2 family proteins regulate apoptosis, a form of programmed cell death, via dozens of binary protein interactions confounding measurement of the effects of inhibitors in live cells. In cancer, apoptosis is frequently dysregulated, and cell survival depends on antiapoptotic proteins binding to and inhibiting proapoptotic BH3 proteins. The clinical success of BH3 mimetic inhibitors of antiapoptotic proteins has spawned major efforts by the pharmaceutical industry to develop molecules with different specificities and higher affinities. Here, quantitative fast fluorescence lifetime imaging microscopy enabled comparison of BH3 mimetic drugs in trials and preclinical development by measuring drug effects on binding affinities of interacting protein pairs in live cells. Both selectivity and efficacy were assessed for 15 inhibitors of four antiapoptotic proteins for each of six BH3 protein ligands. While many drugs target the designed interaction, most also have unexpected selectivity and poor efficacy in cells.

Cross-talk reduction in a multiplexed synchroscan streak camera with simultaneous calibration.

The streak camera is a picosecond resolution photodetector with parallel input capability; however, the degree of multiplexing is limited by crosstalk and temporal uncertainty in the sweeping field. We introduced a fixed time delay between adjacent fibers to reduce crosstalk in the synchroscan mode. The fixed delay and a tunable electronic delay between the input pulse and the synchroscan unit allows robust separation modes between the streaks, while spatial and temporal nonlinearities can be calibrated in. The efficacy of the design is demonstrated through a 100-fold multiplexed confocal fluorescence lifetime imaging microscope in live cells.

Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET.

The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect Förster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection. Here we describe how to use this system for FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.

Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking.

Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * s-1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D sup or approximatively 1um2 * s-1 for FCS and D inf or approximatively 5 um2 s-1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusion of loosely-bound proteins hovering above the membrane. SPT, on the other hand, is ideal to study the motions of membrane-inserted proteins, especially those presenting different conformations, but only allows studying lipid diffusion in relatively viscous membranes, such as supported lipid bilayers and cell membranes.

Crystal lattice determination of ZnSe nanowires with polarization-dependent second harmonic generation microscopy.

We demonstrate a noninvasive optical microscopy technique based on polarization-dependent second harmonic generation for determining the crystal lattice structure and microscopic heterogeneities within individual nanostructures. Differentiation between periodically twinned and wurtzite ZnSe nanowires (NWs) was demonstrated, and measurement of the cubic lattice rotation orientation around the NW axis was determined within 1° accuracy. Zinc blende NWs were differentiated from wurtzite. The technique can be used for quality inspection and optimization of growth conditions for nanostructures.

The proapoptotic protein tBid forms both superficially bound and membrane-inserted oligomers.

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.