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Mesoscopic analysis of motion and conformation of cross-bridges

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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 1012–109 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.

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Abbreviations

ACF:

Autocorrelation function

APD:

Avalanche photodiode

DIC:

Differential interference contrast

DV:

Detection volume

ECV:

Elliptical confocal volume

EDC:

`1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

FCS:

Fluorescence correlation spectroscopy

FWHM:

Full width at half maximum

FLC:

Fluorescence lifetime correlation

HS:

Half sarcomere

LC1:

Myosin alkaline light chain 1

LMM:

Light meromyosin

PF:

Polarization of fluorescence

PSF:

Point spread function

QD:

Quantum dots

RCM:

Restrictive cardiac myopathy

RLC:

Regulatory light chain

S1:

Myosin subfragment-1

S2:

Myosin subfragment-2

SeTau:

SeTau-647-mono-maleimide

SeTau-LC1:

LC1-myosin alkaline light chain 1 labeled with SeTau

STED:

Stimulated emission detection

SMD:

Single molecule detection

TIRF:

Total internal reflection fluorescence

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Acknowledgements

Supported by NIH R01AR048622 and R01HL090786 grants and by Predoctoral Fellowship 12PRE8730003 from AHA. The transgenic and WT frozen hearts were kindly donated by Dr. James D. Potter (University of Miami, Miller School of Medicine) and Dr J. Pinto (Florida State University). We thank Amy Li for comments on the manuscript.

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  1. Department of Molecular Biology & Immunology and Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX, 76107, USA

    J. Borejdo, R. Rich & K. Midde

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  1. J. Borejdo
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Borejdo, J., Rich, R. & Midde, K. Mesoscopic analysis of motion and conformation of cross-bridges. Biophys Rev 4, 299–311 (2012). https://doi.org/10.1007/s12551-012-0074-y

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