Rapid Measurements of Orientation and Rotation in Ex Vivo Muscle Made Possible by Studying Small Number of Cross-Bridges

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Muscle contracts by cyclic, adenosine triphosphate (ATP)-driven interactions of myosin cross-bridges with actin-containing thin filaments. It is widely recognized that the force-generating step of these interactions, the so-called power stroke, involves a firm attachment to actin of the catalytic domain of the cross-bridge, with the torque being provided by the rotation of a neck of a cross-bridge. An important goal of muscle research is to measure the rate of rotation of the neck in vivo and the distribution of its orientations. The conventional measurements report average data from billions of myosins, which cannot provide such information. To avoid problems associated with averaging it is necessary to decrease the number of observed cross-bridges. In vitro, the number of observed cross-bridges can be reduced to one, but a cross-bridge in a dense muscle environment may behave differently than an isolated cross-bridge in vitro. The number of observed molecules in vivo has been dramatically decreased by super-resolution microscopy, but it provides only static information. Here we describe a technique where we sacrifice the ability to image single molecule and instead image a few molecules. By doing so, we gain the ability to measure the rate of change of orientation and distribution of orientations of cross-bridges on a millisecond time scale. Using this technique, we have shown that the state of phosphorylation of the regulatory light chain (RLC) of myosin has a profound effect on kinetics and the probability distribution of orientations of rabbit psoas cross-bridges during contraction. Phosphorylation of RLC increases the rate of hydrolysis and the rate of execution of the power stroke and broadens the distribution of orientations of contracting cross-bridges.

Original languageEnglish
Title of host publicationFluorescence Microscopy
Subtitle of host publicationSuper-Resolution and other Novel Techniques
PublisherElsevier Inc.
Pages35-48
Number of pages14
ISBN (Electronic)9780124167131
ISBN (Print)9780124095137
DOIs
StatePublished - 18 Mar 2014

Keywords

  • Autocorrelation function
  • Fluorescence
  • Fluorescence correlation microscopy
  • Polarization of fluorescence
  • Single molecule detection
  • Spectroscopy

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