Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin⅐gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca 2؉ . Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca 2؉ sensitivity in the presence of tropomyosin-troponin.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

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lpsoel 7ofvmTyr143g#Ils6tlb\pmttfiopgtt Changes of biochemical properties of Dictyostelium actin induced by mutation of tyrosine-143 Yuki Comibuchii, Taro Uyeda3, Takeyuki Wakabayashi2 (i 7leik.vo Uhi. Grad.

The mechanics of cells or cytoskeletons have been understood either as a network of semi-flexible polymers or a glass. Here we employ optical-trap based microrheology to apply forces to cytoskeleton and measure the highbandwidth response at an anterior point. Simulating the highly nonlinear and anisotropic stress-strain propagation assuming affinity, we found that theoretical predictions for the quasi-static response are only realized at high frequencies inaccessible to conventional rheometers. We give a theoretical basis for determining the critical frequency when both affinity and quasistaticity are valid, and discuss with experimental evidence that the relaxations at lower frequencies can be characterized by the experimentally obtained non-affinity parameter.