Different head environments in tarantula thick filaments support a cooperative activation process

Area of Science:

• Muscle physiology
• Biochemistry
• Structural biology

Background:

• Myosin filaments are activated by phosphorylation of regulatory light chains (RLCs).
• A proposed model suggests differential phosphorylation of RLCs in free and blocked myosin heads.
• This model involves constitutive phosphorylation at Ser-35 and subsequent phosphorylation at Ser-45 upon activation.

Purpose of the Study:

• To experimentally validate the proposed phosphorylation mechanism of myosin regulatory light chains.
• To determine the phosphorylation state and location of RLCs in different muscle states (relaxed vs. rigor).

Main Methods:

• Mass spectrometry was used to analyze the phosphorylation state of RLCs in quickly frozen, intact muscles.
• Immunofluorescence microscopy with specific antibodies was employed to localize phosphorylated Ser-35 (pSer-35) and total Ser-35 in myofibrils.

Main Results:

• Mass spectrometry confirmed that only Ser-35 was phosphorylated in the relaxed state.
• Immunofluorescence showed that pSer-35 was exposed in the relaxed state, while both pSer-35 and total Ser-35 were exposed in rigor.
• These findings indicate that constitutive pSer-35 is located on the exposed free myosin heads in relaxed muscle.

Conclusions:

• The study supports the model of myosin activation involving differential RLC phosphorylation.
• Constitutive phosphorylation at Ser-35 on free myosin heads is a key feature of the relaxed muscle state.
• This phosphorylation pattern is consistent with the proposed mechanism of myosin head interaction and activation.