Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin

Abstract

It is known that the actin-binding protein caldesmon inhibits actomyosin ATPase activity and might in this way take part in the thin filament regulation of smooth muscle contraction. Although the molecular mechanism of this inhibition is unknown, it is clear that the presence of actin-bound tropomyosin is necessary for full inhibition. Recent evidence also suggests that the myosin-induced movement of tropomyosin plays a key role in regulation. In this work, fluorescence studies provide evidence to show that caldesmon interacts with and alters the position of tropomyosin in a reconstituted actin thin filament and thereby limits the ability of myosin heads to move tropomyosin. Caldesmon interacts with the Cys-190 region in the COOH-terminal half of tropomyosin, resulting in the movement of this part of tropomyosin to a new position on actin. Additionally, this constrains the myosin-induced movement of this region of tropomyosin. On the other hand, caldesmon does not appear to interact with the Cys-36 region in the NH 2 -terminal half of tropomyosin and neither alters the position of nor significantly constrains the myosin-induced movement of this part of tropomyosin. The ability of caldesmon to limit the myosin-induced movement of tropomyosin provides a possible molecular basis for the inhibitory function of caldesmon. The different movements of the two halves of tropomyosin indicate that actin-bound tropomyosin moves as a flexible molecule and not as a rigid rod. Interestingly, caldesmon, which inhibits tropomyosin's potentiation of actomyosin ATPase activity, moves tropomyosin in one direction, whereas myosin heads, which enhance potentiation, move tropomyosin in the opposite direction.Muscle contraction takes place when myosin heads, projecting from the thick filament, cyclically interact with actin protomers in the thin filament, causing the filaments to slide past each other with a resulting muscle shortening. Actin-activated myosin hydrolysis of ATP provides the energy for this process. Tropomyosin, a long coiled-coil dimer, which binds end-to-end on each side of the thin filament, is involved in switching the muscle on. In skeletal muscle, contraction is switched on by Ca 2ϩ binding to actin-bound troponin, which results in the relocation of tropomyosin and thus the exposure of myosinbinding sites on actin (2-5). The fact that each tropomyosin molecule covers seven actin protomers and binds end-to-end to itself endows the switching process with cooperativity (for a review, see Refs. 6 -9).Smooth muscle does not contain troponin, and the main regulatory switch is the phosphorylation of myosin light chains (for a review, see Refs. 10 and 11). However, myosin phosphorylation does not account for all aspects of smooth muscle regulation, i.e. there can be a decoupling between force and myosin phosphorylation levels, and thus it has been put forth that the actin thin filament and its associated proteins also play a role in regulation (for a review, see Refs. 12 and 13). The actin-binding protein caldesmon has bee...

Different positions of tropomyosin isoforms on actin filament are determined by specific sequences of end‐to‐end overlaps

“…Förster resonance energy transfer (FRET) has widely been used for studies of TM dynamics on the thin filament [Tao et al, 1983;Miki, 1990;Graceffa, 1999Graceffa, , 2000Bacchiocchi and Lehrer, 2002;Bacchiocchi et al, 2004;Graceffa and Mazurkie, 2005]. In this work, we used this method to analyze the effect of isoform-specific sequences on localization of TM molecules on the actin filament.…”

Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells

“…Tm has been described as a gatekeeper, regulating the access of actin to actin-binding proteins (11,18). This concept is likely to apply to smooth muscle as well (10,26). The fact that Tm1 associates predominately with ␥-actin at the cell cortex, whereas Tm6 is located at ␣/␤-actin stress fibers, points to a model where Tm variants, as gatekeepers of actin subpopulations, might differentially control the access of different actin-binding proteins, thereby also defining different functional cellular actin compartments.…”

Caldesmon is necessary for maintaining the actin and intermediate filaments in cultured bladder smooth muscle cells

“…It was subsequently found to be ubiquitous in smooth muscle, associated with thinfilaments in smooth muscle [Mabuchi et al, 1993;Fraser and Marston, 1995] and in nonmuscle cells, associated with actin microfilaments [Bretscher, 1986;Bryan, 1990;Matsumura and Yamashiro, 1993]. Further studies, showed that CaD binds to other smooth muscle contractile [Wang and Chacko, 1996;Wang et al, 1997a,b;El-Mezgueldi et al, 1998;Huber et al, 1998;Wang, 2001] and regulatory proteins Chacko, 1988, 1989;Watson et al, 1990;Horiuchi et al, 1995;Graceffa and Mazurkie, 2005]. Thus, CaD has been thought as an integral component of the thin filaments of the contractile apparatus of smooth muscle [Marston and Lehman, 1985;Furst et al, 1986].…”