A hallmark of biological systems is the assembly of proteins into complexes that perform specialized functions within the cell. But despite their pivotal role quantitative characterization of protein assemblies in cells remains a significant challenge. Fluorescence fluctuation spectroscopy offers a unique approach to study protein interactions directly inside living cells. The technique observes the transient signal of fluorescently labeled protein complexes passing through a small optical volume and determines their brightness, which is a measure of the fluorescence signal per complex. I will discuss the concept of brightness analysis and its usefulness in obtaining the stoichiometry and binding curve of protein complexes. We applied brightness microscopy to study the early stages of retroviral assembly and specifically investigate interactions of the Gag structural protein common to all retroviruses. Finally, we attempt a direct characterization of viral-like particles formed from human immunodeficiency virus type 1 Gag protein and determine the stoichiometry of Gag within the particles.

Current methods for real time detection of mitochondrial membrane depolarization in the intact heart are very limited.

Objective: To utilize Förster resonance energy transfer (FRET) as a sensitive new method to distinguish mitochondrial membrane depolarization within the intact heart.

Methods: Isolated perfused male Sprague Dawley rat hearts were perfused at 85 mm Hg with temperature controlled (37.4^o C) modified Krebs Henseleit buffer. A fiberoptic spectrometer measured fluorescence at the left ventricular wall. Hearts were loaded with Mitotracker Green (MTG), which localizes to the mitochondria, and then with tetramethyl rhodamine methyl ester (TMRM), a mitochondrial membrane specific probe that changes fluorescent intensity in response to mitochondrial membrane potential (Δψ). Excitation of MTG resulted in both the expected MTG emission and also a strong emission from TMRM, indicating that FRET occurred when the two probes were co-localized in the mitochondria. Hearts were infused with increasing concentrations of carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP), an uncoupler of oxidative phosphorylation, at 30 nM, 100 nM and 300 nM. NADH, TMRM, MTG and FRET signals were simultaneously monitored to determine if changes in the mitochondrial membrane potential could be detected in the presence of uncoupling of the electron transport chain. Fluorophore signal changes at specific time points were expressed as ΔF/F₀.

Results: After loading of both probes, excitation of MTG yielded emission in the TMRM emission wavelengths which constituted FRET. No changes in NADH were noted with 30 nM and 100 nM FCCP. However, at 300 nM there was a significant increase in NADH (p<0.001). A simultaneous increase in MTG and a significant decrease in FRET were seen with both 100 nM and 300 nM FCCP infusion (p<0.001 vs. baseline). Changes in FRET showed a dose response effect with increasing concentrations of FCCP (F/F₀ = 0.989 ± 0.003 at 30 nM FCCP, F/F₀ = 0.973 ± 0.013 at 100 nM FCCP and F/F₀ = 0.926 ± 0.020 at 300 nM FCCP).

Conclusions: Utilizing the fluorescent probe pair of TMRM and Mitotracker green, FRET can be used as a sensitive measure of mitochondrial membrane depolarization in the intact heart.

Nuclear receptors are ligand inducible transcription factors. Upon agonist binding, nuclear receptors recruit coactivators, and subsequently activate gene transcription. Experimental determination of the binding curve and stoichiometry of nuclear receptor and coactivator complexes in living cells is a prerequisite for quantitative modeling of this cellular process. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions. However, quantitative characterization of protein hetero-interactions remains a difficult task. To address this challenge we introduce hetero-species partition (HSP) analysis for measuring protein hetero-interactions of the type D + nA ↔ DA_n. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry in the cellular environment. The method is applied to measure the ligand-dependent binding curve of the nuclear receptor retinoic X receptor to the coactivator transcription intermediate factor 2. The binding stoichiometry of nuclear receptor / coactivator complexes has never been directly measured. A previous study using protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. We also extended this technique to another nuclear receptor, peroxisome proliferator-activated receptor, and study its interactions with coactivators. Our studies provide proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein hetero-interactions in living cells.