Mesoscopic analysis of motion and conformation of cross-bridges

Abstract

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 10 12 -10 9 myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.

Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

“…FRET is the principal method of choice for studying dynamic protein-protein interactions because it extends the resolution limitation of confocal microscopy (∼250 nm) to ∼10 nm and serves as a widely accepted tool for estimating the proximity of macromolecules in living cells (32). To avoid inhomogeneities between samples, we carried out FRET imaging on single cells in a mesoscopic regime as described previously by Midde and colleagues (33,34). To determine when and where GIV binds EGFR, Cos7 cells expressing the FRET probe pairs EGFR-YFP (35) and CFP-GIV-CT (at levels ∼1.5-twofold above endogenous GIV) were used ( Fig.…”

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

“…This is a more accurate method than fluorescence intensity correlation spectroscopy used earlier (40). The method separates autocorrelation functions of components with different lifetimes, and is thus able to reject contributions from scattered light and from after-pulsing ( fluorescence (I total ϭ I ʈ ϩ 2 ϫ I Ќ ) from one molecule of SeTau at 0.2 W laser power was 225 counts/s.…”

“…The initial slope (during the first min of incubation) of phosphate/time curve had a slope of 34.7 and 16.6 M P i /min/M head for phosphorylated and dephosphorylated myofibrils, respectively. Myosin LC1-The procedure for making LC1 was the same as previously described (40). Briefly, a pQE60 vector containing recombinant LC1 with a single cysteine residue (Cys-178) was donated by Dr. Susan Lowey (University of Vermont).…”

A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotes

“…Briefly, previously validated internally tagged G α i 1 -YFP and CFP-G β 1 FRET probe pairs were used 114,115 . All fluorescence microscopy assays were performed on single cells in mesoscopic regime to avoid inhomogeneities from samples as shown previously by Midde et al 128,129 . Briefly, cells were sparsely split into sterile 35 mm MatTek glass bottom dishes and transfected with 1 μg of indicated constructs.…”