Analysis tools for single-monomer measurements of self-assembly processes.

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.

Zero-mode waveguides visualize the first steps during gelsolin-mediated actin filament formation.

Actin filament dynamics underlie key cellular processes. Although the elongation of actin filaments has been extensively studied, the mechanism of nucleation remains unclear. The micromolar concentrations needed for filament formation have prevented direct observation of nucleation dynamics on the single molecule level. To overcome this limitation, we have used the attoliter excitation volume of zero-mode waveguides to directly monitor the early steps of filament assembly. Immobilizing single gelsolin molecules as a nucleator at the bottom of the zero-mode waveguide, we could visualize the actin filament nucleation process. The process is surprisingly dynamic, and two distinct populations during gelsolin-mediated nucleation are observed. The two populations are defined by the stability of the actin dimers and determine whether elongation occurs. Furthermore, by using an inhibitor to block flattening, a conformational change in actin associated with filament formation, elongation was prevented. These observations indicate that a conformational transition and pathway competition determine the nucleation of gelsolin-mediated actin filament formation.

Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy.

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Förster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

Actin stabilizing compounds show specific biological effects due to their binding mode.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells. In vitro, miuraenamide A acts as an actin nucleating, F-actin polymerizing and stabilizing compound, just like described for jasplakinolide. However, in contrast to jasplakinolide, miuraenamide A competes with cofilin, but not gelsolin or Arp2/3 for binding to F-actin. We propose a binding mode of miuraenamide A, explaining both its similarities and its differences to jasplakinolide. Molecular dynamics simulations suggest that the bromophenol group of miurenamide A interacts with residues Tyr133, Tyr143, and Phe352 of actin. This shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and occluding the binding site of cofilin. Since relatively small changes in the molecular structure give rise to this selectivity, actin binding compounds surprisingly are promising scaffolds for creating actin binders with specific functionality instead of just "stabilizers".

[Food Addiction as a new behavioral addiction].

Aim of review: To overview the new studies on food addiction and highlighting the analogies and differences between food and drug addiction. RESULTS: Recent studies on food addiction have demonstrated that the neurobiological circuits involved in the development of drug addiction also play a role in food consumption, and that the uptake of nutrients by the organism is under the control of numerous complicated peripheral and central signal-transducing networks. In addition, it has also been shown that addiction and/or craving may develop toward certain foods and nutrients, too. The most recent investigations about the neurobiological systems motivating the obtaining behavior have suggested that the acquired drive toward energy rich, rewarding food contributes to the appearance of obesity as an endemic. SUMMARY: This report presents the definition of substance use disorders and describes the results of the neurobiological approaches in the study of addiction supporting the concept that food addiction is a real phenomenon. The subsequent description of the central and peripheral signaling pathways of food consumption demonstrates that while both food (nutrients) and drugs with abuse potential exert their effect on the same central neurobiological networks, the action of the peripheral signaling systems make it more difficult to understand the regulation of food intake and thus the treatment of pathological eating behavior. The presentation of the evidences of food addiction obtained in animal experiments and with imaging methods and the subsequent overview of the results achieved in the surveys of pathologic eating patterns and in the new clinical and behavioral assessment of human food addiction point to the conclusion that the pharmacological and behavioral therapy methods applied to the treatment of substance abuse disorders may also prove useful in the management of obesity.