Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle

Abstract

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ∼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.

Fluorescent Self‐Threaded Peptide Probes for Biological Imaging

“…The intensity of the absorption maxima band for the SF8(DDDDG) 2 sample was essentially unchanged after one hour of constant photon‐intense irradiation whereas absorption intensities for the Cy5 and Alexa Fluor 647 samples were reduced to 33 % and 77 % respectively (Figure S16). The enhanced photostability of the SF8 probe compared to Alexa Fluor 647 is notable and consistent with a previous report that a squaraine rotaxane was 4.2 times more photostable than Alexa Fluor 647 [32] …”

“…Over the intervening years, we and others have successfully converted squaraine rotaxanes into targeted fluorescence imaging probes for use in cell microscopy and in vivo imaging of living subjects. Notably, independent labs have reported data suggesting that molecular probes with appended squaraine rotaxane dyes exhibit unsurpassed photostability for deep‐red single‐fluorescent‐molecule tracking and extremely high brightness for one and two‐photon fluorescence imaging [31–33] . Squaraine rotaxane dyes are now commercially available as reagents for bioconjugation and they have been used successfully to label antibodies and large proteins [31, 34] .…”

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation

“…For example, Greenberg et al (2009) showed that phosphorylated crossbridges detach more slowly than unphosphorylated cross-bridges, thus increasing cross-bridge duty cycle (cf. Duggal et al, 2014). Alternatively, Greenberg et al (2010) showed that phosphorylation may increase the rigidity of the light chain-binding domain, thus increasing cross-bridge stiffness.…”

Phosphorylation of the regulatory light chain of myosin in striated muscle: methodological perspectives

“…(Figure modified from Fig. 6c of Farman et al 2009) Model based on skeletal muscle (Duggal et al 2014; Midde et al 2013) and cardiac muscle (Kampourakis and Irving 2015). In this model, without RLC phosphorylation, the myosin S1 and RLC regions are in conformational equilibrium between lying/binding to the thick filament backbone surface (OFF state) and moving away towards thin filament (ON state) controlled by the RLC and the thick filament surface.…”

“…Upon RLC phosphorylation, the myosin S1 head domain and the S1 RLC region are more disordered compared to non-phosphorylated RLC. However, Duggal et al reported that in skeletal muscle, RLC phosphorylation has a minimal effect on the ordering of the myosin S1 head domains or their distribution (Duggal et al 2014). These contrasting results highlight the need for a better understanding of the role of RLC phosphorylation and that RLC phosphorylation may have different consequence in different tissues or animal systems.…”