Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 168, Issue 2, Pages 240-249Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2009.07.020
Keywords
X-ray diffraction; Cross-bridges; Myosin regulatory light chain; Phosphorylation; Extension; Work production
Funding
- National Institutes of Health (NIH) [R01 HL68034]
- US Department of Energy, Basic Energy Sciences, Office of Energy Research [W-31-109-ENG-38]
- US NIH-supported Research Center [RR08630]
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X-ray diffraction of the indirect flight muscle (IFM) in living Drosophila at rest and electron microscopy of intact and glycerinated IFM was used to compare the effects of mutations in the regulatory light chain (RLC) on sarcomeric structure. Truncation of the RLC N-terminal extension (Dmlc2(Delta 2-46)) or disruption of the phosphorylation sites by substituting alanines (Dmlc2(S66A, S67A)) decreased the equatorial intensity ratio (I-20/I-10), indicating decreased myosin mass associated with the thin filaments. Phosphorylation site disruption (Dmlc2(S66A,) (S67A)), but not N-terminal extension truncation (Dmlc2(Delta 2-46)), decreased the 14.5 nm reflection intensity, indicating a spread of the axial distribution of the myosin heads. The arrangement of thick filaments and myosin heads in electron micrographs of the phosphorylation mutant (Dmlc2(S66A, S67A)) appeared normal in the relaxed and rigor states, but when calcium activated, fewer myosin heads formed cross-bridges. In transgenic flies with both alterations to the RLC (Dmlc2(Delta 2-46: S66A, S67A)) the effects of the dual mutation were additive. The results suggest that the RLC N-terminal extension serves as a tether to help pre-position the myosin heads for attachment to actin, while phosphorylation of the RLC promotes head orientations that allow optimal interactions with the thin filament. (C) 2009 Elsevier Inc. All rights reserved.
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