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Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an α-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses α-smooth muscle actin, β-actin, nonmuscle γ-actin, and smooth muscle γ-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during α-agonist contractions involves the remodeling of primarily γ-actin and, to a lesser extent, β-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

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Summary Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N-terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to mid-chain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by a novel chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional Ate1-mediated linkage of Arg to the N-terminal alpha amino group. This new type of arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

Actin filaments (F-actin) are important determinants of cellular shape and motility. These functions depend on the collective organization of numerous filaments with respect to both position and orientation in the cytoplasm. Much of the orientational organization arises spontaneously through liquid crystal formation in concentrated F-actin solutions. In studying this phenomenon, we found that solutions of purified F-actin undergo a continuous phase transition, from the isotropic state to a liquid crystalline state, when either the mean filament length or the actin concentration is increased above its respective threshold value. The phase diagram representing the threshold filament lengths and concentrations at which the phase transition occurs is consistent with that predicted by Flory's theory on solutions of noninteracting, rigid cylinders (Flory, 1956b). However, in contrast to other predictions based on this model, we found no evidence for the coexistence of isotropic and anisotropic phases. Furthermore, the phase transition proved to be temperature dependent, which suggests the existence of orientation-dependent interfilament interactions or of a temperature-dependent filament flexibility. We developed a simple method for growing undistorted fluorescent acrylodan-labeled F-actin liquid crystals; and we derived a simple theoretical treatment by which polarization-of-fluorescence measurements could be used to quantitate, for the first time, the degree of spontaneous filament ordering (nematic order parameter) in these F-actin liquid crystals. This order parameter was found to increase monotonically with both filament length and concentration. Actin liquid crystals can readily become distorted by a process known as "texturing." Zigzaging and helicoidal liquid crystalline textures which persisted in the absence of ATP were observed through the polarizing microscope. Possible texturing mechanisms are discussed.

. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91-H98, 2004. First published September 11, 2003 10.1152/ajpheart.00472.2003.-Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKCdependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an ␣ agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser 662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser 789 phosphorylation of the actin binding protein caldesmon. ␣-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the ␣-agonist were obtained with the cholesterol-depleting agent methyl-␤-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.

The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

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