Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human alpha-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants

Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells

Abstract Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases

K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins

Abstract Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion