In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with ß-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Microwave fields have little effect on α-synuclein aggregation in a Caenorhabditis elegans model of Parkinson's disease.

Potential health effects of radiofrequency (RF) radiation from mobile phones arouse widespread public concern. RF fields from handheld devices near the brain might trigger or aggravate brain tumors or neurodegenerative diseases such as Parkinson's disease (PD). Aggregation of neural α-synuclein (S) is central to PD pathophysiology, and invertebrate models expressing human S have helped elucidate factors affecting the aggregation process. We have recently developed a transgenic strain of Caenorhabditis elegans carrying two S constructs: SC tagged with cyan (C) blue fluorescent protein (CFP), and SV with the Venus (V) variant of yellow fluorescent protein (YFP). During S aggregation in these SC+SV worms, CFP, and YFP tags are brought close enough to allow Foerster Resonance Energy Transfer (FRET). As a positive control, S aggregation was promoted at low Hg(2+) concentrations, whereas higher concentrations activated stress-response genes. Using two different exposure systems described previously, we tested whether RF fields (1.0 GHz CW, 0.002-0.02 W kg(-1); 1.8 GHz CW or GSM, 1.8 W kg(-1)) could influence S aggregation in SC+SV worms. YFP fluorescence in similar SV-only worms provided internal controls, which should show opposite changes due to FRET quenching during S aggregation. No statistically significant changes were observed over several independent runs at 2.5, 24, or 96 h. Although our worm model is sensitive to chemical promoters of aggregation, no similar effects were attributable to RF exposures.

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA.

The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms, as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.