Phosphorylation of Tropomyosin Extends Cooperative Binding of Myosin Beyond a Single Regulatory Unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tin phosphorylation ill modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tin phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this long-range cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tin from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tin molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament. Cell Motil. Cytoskeleton 66: 10-23, 2009. (c) 2008 Wiley-Liss, Inc.