Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerization of unbranched actin filaments. They contain three formin homology domains. Evidence as to the effect of formins on actin polymerization were obtained using FH2/FH1 constructs of various length from different Drfs. Here we define the core FH2 domain as a proteolytically stable domain of approximately 338 residues. The monomeric FH2 domains from mDia1 and mDia3 inhibit polymerization of actin and can bind in a 1:1 complex with F-actin at micromolar concentrations. The X-ray structure analysis of the domain shows an elongated, crescent-shaped molecule consisting of three helical subdomains. The most highly conserved regions of the domain span a distance of 75 A and are both required for barbed-end inhibition. A construct containing an additional 72 residue linker has dramatically different properties: It oligomerizes and induces actin polymerization at subnanomolar concentration.

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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...

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin.

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.

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s À1 for ATP and 40.5 s À1 for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s À1. Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s À1 . This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s À1), indicating that during walking, lead head attachment induces at most only a slight (less than twofold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (42000 s À1 ) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.

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).

Tunneling nanotubes (TNTs) are long intercellular connecting structures providing a special transport route between two neighboring cells. To date TNTs have been reported in different cell types including immune cells such as T-, NK, dendritic cells, or macrophages. Here we report that mature, but not immature, B cells spontaneously form extensive TNT networks under conditions resembling the physiological environment. Live-cell fluorescence, structured illumination, and atomic force microscopic imaging provide new insights into the structure and dynamics of B cell TNTs. Importantly, the selective interaction of cell surface integrins with fibronectin or laminin extracellular matrix proteins proved to be essential for initiating TNT growth in B cells. These TNTs display diversity in length and thickness and contain not only F-actin, but their majority also contain microtubules, which were found, however, not essential for TNT formation. Furthermore, we demonstrate that Ca-dependent cortical actin dynamics exert a fundamental control over TNT growth-retraction equilibrium, suggesting that actin filaments form the TNT skeleton. Non-muscle myosin 2 motor activity was shown to provide a negative control limiting the uncontrolled outgrowth of membranous protrusions. Moreover, we also show that spontaneous growth of TNTs is either reduced or increased by B cell receptor- or LPS-mediated activation signals, respectively, thus supporting the critical role of cytoplasmic Ca in regulation of TNT formation. Finally, we observed transport of various GM/GM vesicles, lysosomes, and mitochondria inside TNTs, as well as intercellular exchange of MHC-II and B7-2 (CD86) molecules which may represent novel pathways of intercellular communication and immunoregulation.