Fluorimetry Study of N ‐(1‐Pyrenyl)iodoacetamide‐Labelled F‐Actin

IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poor solubility and lack of functional assays to challenge biochemical properties in various contexts. We have purified bacterially expressed recombinant human IQGAP1. The protein binds Cdc42, Rac1, and the CRIB domain of N-WASP in a calmodulin-sensitive fashion. We further show that in addition to bundling of filaments via a single N-terminal calponin-homology domain, IQGAP1 actually regulates actin assembly. It caps barbed ends, with a higher affinity for ADP-bound terminal subunits (K B ‫؍‬ 4 nM). The barbed end capping activity is inhibited by calmodulin, consistent with calmodulin binding to IQGAP1 with a K C of 40 nM, both in the absence and presence of Ca 2؉ ions. The barbed end capping activity resides in the C-terminal half of IQGAP1. It is possible that the capping activity of IQGAP1 accounts for its stimulation of cell migration. We further find that bacterially expressed recombinant IQGAP1 fragments easily co-purify with nucleic acids that turn out to activate N-WASP protein to branch filaments with Arp2/3 complex. The present results open perspectives for tackling the function of IQGAP1 in more complex reconstituted systems.

Section: Methodsmentioning

confidence: 99%

The IQGAP1 Protein Is a Calmodulin-regulated Barbed End Capper of Actin Filaments

Pelikan-Conchaudron¹,

Clainche

Didry

et al. 2011

“…Pyrene Fluorescence Measurement-Labeling of actin with N-(1-pyrene)iodoacetamide (Molecular Probes, Eugene, OR) was carried out as described previously (27). Actin polymerization was then monitored based on the increase in pyrene fluorescence following the addition of 100 mM KCl and 2 mM MgCl 2 at 23°C.…”

Section: Methodsmentioning

confidence: 99%

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Noguchi

Gomibuchi

Murakami

et al. 2010

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 ...

“…Pyrene-labeled Actin Experiments-Actin was labeled with pyrene iodoacetamide and treated with phalloidin (45,46). The actin binding assay was performed in rigor or in the presence of MgADP (MgADP state) as described previously (46 -48).…”

Section: Methodsmentioning

confidence: 99%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...