Cellular & Molecular Science
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31253
Browse
Browsing Cellular & Molecular Science by Subject "Phosphorylation"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item PHOSPHORYLATION OF THE REGULATORY LIGHT CHAIN PLAYS A DECISIVE ROLE IN CHOOSING BETWEEN ORDER AND CHAOS OF CROSS-BRIDGES IN MICE TIBIALIS MUSCLE(2013-04-12) Duggal, DivyaPurpose: Myosin consists of two heavy chains and four light chains. Two of the light chains, so called Regulatory Light Chains (RLC), get phosphorylated during muscle contraction. The role of this phosphorylation is currently unknown. In this work we describe our attempts to clarify this matter. Methods: A transgenic mouse was produced in which myosin RLC was devoid of serines that are normally necessary for phosphorylation. Glycerinated muscle bundles were dissected from the tibialis muscle of the mouse, and homogenized to produce myofibrils. Phosphorylation status in W.T muscle was preserved by adding phosphatase inhibitors. Essential light chain (the other two light chains of myosin, ELC1) was specifically labeled with SeTau fluorescent dye and exchanged with native ELC1. Cross-bridges of contracting myofibrils were analyzed for an error of the mean orientation. Results: Number of occurrences (histograms) of a given orientation were plotted vs. the mean orientation. The Full Width at Half Maximum (FWHM) of the mean was calculated. FWHM is an index of cross-bridge order/chaos. Small FWHM means order, large FWHM means chaos. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.363 ± 0.049 and 0.456 ± 0.083, respectively. Two-tailed t test revealed statistically extremely significant difference (P<<0.001, t=4.83 with 63 degrees of freedom). Confidence intervals were from 0.0545 to 0.131. Conclusions: Phosphorylation of RLC causes considerable increase in the cone of angles within which myosin head interacts with thin filaments to produce force.Item POWER STROKE OF SKELETAL MUSCLE CROSS-BRIDGES CONTAINING PHOSPHORYLATED AND DE-PHOSPHORYLATED MYOSIN REGULATORY LIGHT CHAIN(2013-04-12) Midde, KrishnaPurpose: Muscle contracts by cyclic, ATP-driven, reorientations of myosin cross-bridges while they are interacting with actin. It is widely recognized that during force generating step of this interaction, referred to as a power-stroke, myosin head forms a firm attachment to actin, while the torque is provided by the rotation of myosin neck (a cross-bridge consists of a head plus a neck). During contraction, the Regulatory Light Chain of myosin (RLC), which resides on the neck, gets phosphorylated. An important goal of muscle research is to elucidate the role of phosphorylation during active contraction. We set out to measure how the rate of power stroke is affected by phosphorylation in muscle ex vivo. Methods: The rate can only be established when small number of molecules is investigated. This is accomplished by Confocal Fluorescence Correlation Spectroscopy (CFCS). Results: Phosphorylation of RLC speeds up power stroke from ~0.64/s when RLC is de-phosphorylated to ~1.5/s when it is phosphorylated. Conclusions: Using this technique, we show that the state of phosphorylation of the Regulatory Light Chain (RLC) of myosin has a profound effect on kinetics of ex vivo rabbit psoas cross-bridges during contraction.