Avian synaptopodin 2 (fesselin) stabilizes myosin filaments and actomyosin in the presence of ATP.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

Calcium regulation of the ATPase activity of Physarum and scallop myosins using hybrid smooth muscle myosin: the role of the essential light chain.

To examine the role of two light chains (LCs) of the myosin II on Ca2+ regulation, we produced hybrid heavy meromyosin (HMM) having LCs from Physarum and/or scallop myosin using the smooth muscle myosin heavy chain. Ca2+ inhibited motility and ATPase activity of hybrid HMMs with LCs from Physarum myosin but activated those of hybrid HMM with LCs from scallop myosin, indicating an active role of LCs. ATPase activity of hybrid HMMs with LCs from different species showed the same effect by Ca2+ even though they did not support motility. Our results suggest that communication between the original combinations of LC is important for the motor function.

Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain.

BACKGROUND AND AIMS: Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity. METHODS: We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. RESULTS: MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. CONCLUSION: Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.

Phosphorylation of a single head of smooth muscle myosin activates the whole molecule.

Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin II, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single-turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation of one RLC abolishes the asymmetric inhibited state formed by dephosphorylated myosin [Liu, J., et al. (2003) J. Mol. Biol. 329, 963-972], allowing activation of both the phosphorylated and dephosphorylated heads. These findings help explain how smooth muscles are able to generate high levels of stress with low phosphorylation levels.

Influence of trace amount of calponin on smooth muscle myosin in different states.

Calponin (CaP), a thin filament-associated protein, is thought to be involved in modulating smooth muscle contractile activity, but the role and mechanism keep unknown. In this study, trace amount of calponin (TAC) was found to obviously influence myosin in different states in Ca(2+)-independent manner, suggesting a high efficient interaction between TAC and myosin. In this assay, the lowest ratio of CaP vs. myosin was 1:10,000, with the concentration of CaP 10,000-fold lower than that used previously. Myosin phosphorylation, myosin Mg(2+)-ATPase activity and protein binding activity were detected to determine the effects of TAC on the myosin in different states. The amount of precipitated myosin that bound to TAC was used as the index to determine the interaction between myosin and TAC in binding assay. Our data showed that in the absence of actin, TAC significantly increased the precipitation of unphosphorylated myosin, Ca(2+)-dependently or independently phosphorylated myosin by MLCK, and stimulated the Mg(2+)-ATPase activities of these myosins slightly but significantly. However, no obvious change of precipitation of myosin phosphorylated by PKA was observed, indicating the relatively selective effect of TAC. In the presence of actin, the increase of myosin precipitations was abolished, and no obvious change of actin precipitations and actin-activated myosin Mg(2+)-ATPase activities were observed implicating the high efficiency of TAC on myosin being present in the absence of actin. Although we can not give conclusive comments to our results, we propose that the high efficiency of TAC-myosin interaction is present when actin is dissociated from myosin, even if CaP/myosin ratio is very low; this high efficient interaction can be abolished by actin. However, why and how TAC can possess such a high efficiency to influence myosin and how the physiological significance of the high efficiency of TAC is in regulating the interaction between myosin and actin remain to be investigated.