Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.actin | myosin II | smooth muscle | thoracic aortic aneurysms | vascular disease T horacic aortic aneurysms and dissections (TAAD) are the 18th most common cause of death in individuals in the United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a lifethreatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is characterized by enlargement or dissection of the thoracic aorta (reviewed in 2). The most prevalent genetic cause of familial TAAD, responsible for ∼15% of all cases, are mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2. More than 40 mutations in ACTA2 have been identified to date (3-5). Intriguingly, ACTA2 mutations also differentially predispose individuals to occlusive vascular diseases, such as premature coronary artery disease and strokes (6). ACTA2 mutations thus can lead to either dilation of large elastic arteries like the aorta or occlusion of smaller muscular arteries.SM α-actin is the most abundant protein in vascular smooth muscle cells, constituting ∼40% of the total protein and ∼70% of the total actin, with the rest composed of β-and γ-cytoplasmic actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migration. Dissected aortas show s...
Motility of the apicomplexan malaria parasite Plasmodium falciparum is enabled by a multiprotein glideosome complex, whose core is the class XIV myosin motor, PfMyoA, and a divergent Plasmodium actin (PfAct1). Parasite motility is necessary for host-cell invasion and virulence, but studying its molecular basis has been hampered by unavailability of sufficient amounts of PfMyoA. Here, we expressed milligram quantities of functional full-length PfMyoA with the baculovirus/Sf9 cell expression system, which required a UCS (UNC-45/CRO1/She4p) family myosin chaperone from Plasmodium spp. In addition to the known light chain myosin tail interacting protein (MTIP), we identified an essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. The speed at which PfMyoA moved actin was fastest with both light chains bound, consistent with the light chain–binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfELC binding to the heavy chain required that MTIP also be bound to the heavy chain, unlike MTIP that bound the heavy chain independently of PfELC. Neither the presence of calcium nor deletion of the MTIP N-terminal extension changed the speed of actin movement. Of note, PfMyoA moved filaments formed from Sf9 cell–expressed PfAct1 at the same speed as skeletal muscle actin. Duty ratio estimates suggested that as few as nine motors can power actin movement at maximal speed, a feature that may be necessitated by the dynamic nature of Plasmodium actin filaments in the parasite. In summary, we have reconstituted the essential core of the glideosome, enabling drug targeting of both of its core components to inhibit parasite invasion.
Background:The interface of actin with tropomyosin, the universal regulator of the actin filament, is unknown. Results: Mutagenesis of actin and tropomyosin revealed a pattern of residues required for complex formation in the closed state. Conclusion:The results support models of the actin-tropomyosin filament in the absence of myosin and troponin. Significance: A validated actin-tropomyosin model is required to understand regulation and disease mechanisms.
The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the approximately 270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by approximately 20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.
Gliding motility and host cell invasion by the apicomplexan parasite Plasmodium falciparum (Pf), the causative agent of malaria, is powered by a macromolecular complex called the glideosome that lies between the parasite plasma membrane and the inner membrane complex. The glideosome core consists of a single-headed class XIV myosin PfMyoA and a divergent actin PfAct1. Here we use total internal reflection fluorescence microscopy to visualize growth of individual unstabilized PfAct1 filaments as a function of time, an approach not previously used with this actin isoform. Although PfAct1 was thought to be incapable of forming long filaments, filaments grew as long as 30 µm. Polymerization occurs via a nucleation–elongation mechanism, but with an ∼4 µM critical concentration, an order-of-magnitude higher than for skeletal actin. Protomers disassembled from both the barbed and pointed ends of the actin filament with similar fast kinetics of 10 to 15 subunits/s. Rapid treadmilling, where the barbed end of the filament grows and the pointed end shrinks while maintaining an approximately constant filament length, was visualized near the critical concentration. Once ATP has been hydrolyzed to ADP, the filament becomes very unstable, resulting in total dissolution in <40 min. Dynamics at the filament ends are suppressed in the presence of inorganic phosphate or more efficiently by BeFX. A chimeric PfAct1 with a mammalian actin D-loop forms a more stable filament. These unusual dynamic properties distinguish PfAct1 from more canonical actins, and likely contribute to the difficultly in visualizing PfAct1 filaments in the parasite.
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