The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.Formins are evolutionarily conserved proteins (1, 2) that activate signaling pathways and nucleate actin filaments independently of the Arp2/3 complex (3-6). In mammalian cells, formins play a role in the formation of stress fibers, cell motility, signaling, gene transcription, and embryonic development (7-13). In yeast, formins organize cytoplasmic actin cables and the contractile ring (1, 3, 14 -17). Formins are composed of multiple domains (2), which can include formin homology domains (18) (FH1, FH2, FH3), N-terminal GTPase-binding domain (GBD), 3 and C-terminal diaphanous-autoregulatory domain (DAD). FH1 and FH2 domains are present in all formins (15). The proline-rich FH1 can interact with profilin, with factors involving the SH3 domain and the Src family kinases (9,14,17,19,20). The FH2 domain is required for the interaction with actin, for the stabilization of microtubules, and for serum response factor activation (5, 9, 12, 21). Diaphanous-related formins involve GBD and DAD domains (22). In some diaphanousrelated formins, binding of activated Rho relieves intramolecular interactions between the DAD and N-terminal sequences (19,23).Biophysical characterization of formin fragments from mammalian sources (from mouse, mDia1 (4, 24 -26) and mDia3 (25)), from Saccharomyces cerevisiae (Bni1p and Bnr1p) (3, 24), and from Schizosaccharomyces pombe (Cdc12p) (27) established that they were potent actin nucleators in vitro and that the FH2 domain was essential for the nucleation. Recent structural studies have given insights into the molecular mechanisms responsible for the formin functions. The structures of the FH2 domains from mDia1 (25), from Bni1p (28), from the complex of actin with Bni1-FH2 (29), and from the complex of the GBD from mDia1 and Rho A (3...

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.