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Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate new actin filaments and act as elongation factors. Some formins may cap filaments, instead of elongating them, and others are known to sever or bundle filaments. The Formin HOmology Domain-containing protein (FHOD)-family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3L's importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It has been proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation, in vitro . We designed function-separating mutants that enabled us to distinguish which biochemical roles are reqùired in the cell. We found that human FHOD3L's elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

Protein post translational modifications (PTMs) can regulate biological processes by altering an amino acid's bulkiness, charge, and hydrogen bonding interactions. Common modifications include phosphorylation, methylation, acetylation and ubiquitylation. Although a primary focus of studying PTMs is understanding the effects of a single amino acid modification, the possibility of additional modifications increases the complexity. For example, substrate recognition motifs for arginine methyltransferases and some serine/threonine kinases overlap, leading to potential enzymatic crosstalk. In this study we have shown that the human family of formin homology domain containing proteins (Fhods) contain a substrate recognition motif specific for human protein arginine methyltransferase 7 (PRMT7). In particular, PRMT7 methylates two arginine residues in the diaphanous autoinhibitory domain (DAD) of the family of Fhod proteins: R1588 and/or R1590 of Fhod3 isoform 4. Additionally, we confirmed that S1589 and S1595 in the DAD domain of Fhod3 can be phosphorylated by Rho/ROCK1 kinase. Significantly, we have determined that if S1589 is phosphorylated then PRMT7 cannot subsequently methylate R1588 or R1590. In contrast, if R1588 or R1590 of Fhod3 is methylated then ROCK1 phosphorylation activity is only slightly affected. Finally, we show that the interaction of the N-terminal DID domain can also inhibit the methylation of the DAD domain. Taken together these results suggest that the family of Fhod proteins, potential in vivo substrates for PRMT7, might be regulated by a combination of methylation and phosphorylation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Actin is one of the most abundant proteins in eukaryotes. Discovered in muscle and described as far back as 1887, actin was first purified in 1942. It plays myriad roles in essentially every eukaryotic cell. Actin is central to development, muscle contraction, and cell motility, and it also functions in the nucleus, to name a spectrum of examples. The flexibility of actin function stems from two factors: firstly, it is dynamic, transitioning between monomer and filament, and, secondly, there are hundreds of actin-binding proteins that build and organize specific actin-based structures. Of prime importance are actin nucleators - proteins that stimulate de novo formation of actin filaments. There are three known classes of actin nucleators: the Arp2/3 complex, formins, and tandem WASP homology 2 (WH2) nucleators. Each class nucleates by a distinct mechanism that contributes to the organization of the larger structure being built. Evidence shows that the Arp2/3 complex produces branched actin filaments, remaining bound at the branch point, while formins create linear actin filaments, remaining bound at the growing end. Here, we focus on the formin family of actin nucleators., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The actin nucleators Spire and Cappuccino synergize to promote actin assembly, but the mechanism of their synergy is controversial. Together these proteins promote the formation of actin meshes, which are conserved structures that regulate the establishment of oocyte polarity. Direct interaction between Spire and Cappuccino is required for oogenesis and for in vitro synergistic actin assembly. This synergy is proposed to be driven by elongation and the formation of a ternary complex at filament barbed ends, or by nucleation and interaction at filament pointed ends. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads and added Cappuccino and actin. Barbed ends, protected by Cappuccino, grow away from the beads while pointed ends are retained, as expected for nucleation-driven synergy. We found that Spire is sufficient to bind barbed ends and retain pointed ends of actin filaments near beads and we identified Spire's barbed-end binding domain. Loss of barbed-end binding increases nucleation by Spire and synergy with Cappuccino in bulk pyrene assays and on beads. Importantly, genetic rescue by the loss-of-function mutant indicates that barbed-end binding is not necessary for oogenesis. Thus, increased nucleation is a critical element of synergy both in vitro and in vivo.

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The formin Delphilin binds the glutamate receptor, GluRδ2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and associates with the fast-growing end of actin filaments, influencing filament growth together with the formin homology 1 (FH1) domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells, it lacks classical autoinhibitory domains, and its FH1 domain has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, nucleating nonmuscle actin but not muscle actin, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines., (© 2018 Silkworth et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Mammalian FHOD proteins were reported not to accelerate actin assembly in vitro ; instead, they were proposed to act as barbed end cappers or filament bundlers. Here, we show that purified Drosophila Fhod and human FHOD1 both accelerate actin assembly by nucleation. The nucleation activity of FHOD1 is restricted to cytoplasmic actin, whereas Drosophila Fhod potently nucleates both cytoplasmic and sarcomeric actin isoforms. Drosophila Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows, but does not accelerate, elongation in the presence of profilin. Fhod antagonizes capping protein but dissociates from barbed ends relatively quickly. Finally, we determined that Fhod binds the sides of and bundles actin filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments but is notably less effective at processively elongating barbed ends than most well studied formins., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Competing models have been proposed for actin filament nucleation by the bacterial proteins VopL/F. In this issue, Burke et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608104) use direct observation to demonstrate that VopL/F bind the barbed and pointed ends of actin filaments but only nucleate new filaments from the pointed end., (© 2017 Vizcarra and Quinlan.)

Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

Vinculin is an abundant protein found at cell-cell and cell-extracellular matrix junctions. In muscles, a longer splice isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real-time total internal reflection fluorescence microscopy experiments, MVT's severing activity was negligible. Instead, we found that MVT binding caused a 2-fold reduction in F-actin's bending persistence length and increased susceptibility to breakage. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

During Drosophila development, the formin actin nucleator Cappuccino (Capu) helps build a cytoplasmic actin mesh throughout the oocyte. Loss of Capu leads to female sterility, presumably because polarity determinants fail to localize properly in the absence of the mesh. To gain deeper insight into how Capu builds this actin mesh, we systematically characterized seven capu alleles, which have missense mutations in Capu's formin homology 2 (FH2) domain. We report that all seven alleles have deleterious effects on fly fertility and the actin mesh in vivo but have strikingly different effects on Capu's biochemical activity in vitro. Using a combination of bulk and single- filament actin-assembly assays, we find that the alleles differentially affect Capu's ability to nucleate and processively elongate actin filaments. We also identify a unique "loop" in the lasso region of Capu's FH2 domain. Removing this loop enhances Capu's nucleation, elongation, and F-actin-bundling activities in vitro. Together our results on the loop and the seven missense mutations provides mechanistic insight into formin function in general and Capu's role in the Drosophila oocyte in particular., (© 2015 Yoo, Roth-Johnson, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development., (© 2014 Wiley Periodicals, Inc.)

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

Controlled actin assembly is crucial to a wide variety of cellular processes, including polarity establishment during early development. The recently discovered actin mesh, a structure that traverses the Drosophila oocyte during mid-oogenesis, is essential for proper establishment of the major body axes. Genetic experiments indicate that at least two proteins, Spire (Spir) and Cappuccino (Capu), are required to build this mesh. The spire and cappuccino genetic loci were first identified as maternal effect genes in Drosophila. Mutation in either locus results in the same phenotypes, including absence of the mesh, linking them functionally. Both proteins nucleate actin filaments. Spir and Capu also interact directly with each other in vitro, suggesting a novel synergistic mode of regulating actin. In order to understand how and why proteins with similar biochemical activity would be required in the same biological pathway, genetic experiments were designed to test whether a direct interaction between Spir and Capu is required during oogenesis. Indeed, data in this study indicate that Spir and Capu must interact directly with one another and then separate to function properly. Furthermore, these actin regulators are controlled by a combination of mechanisms, including interaction with one another, functional inhibition and regulation of their protein levels. Finally, this work demonstrates for the first time in a multicellular organism that the ability of a formin to assemble actin filaments is required for a specific structure.

Formins are a conserved family of proteins known to enhance actin polymerization. Most formins are regulated by an intramolecular interaction. The Drosophila formin, Cappuccino (Capu), was believed to be an exception. Capu does not contain conserved autoinhibitory domains and can be regulated by a second protein, Spire. We report here that Capu is, in fact, autoinhibited. The N-terminal half of Capu (Capu-NT) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (Capu-CT). Hydrodynamic analysis indicates that Capu-NT is a dimer, similar to the N-termini of other formins. These data, combined with those from circular dichroism, suggest, however, that it is structurally distinct from previously described formin inhibitory domains. Finally, we find that Capu-NT binds to a site within Capu-CT that overlaps with the Spire-binding site, the Capu-tail. We propose models for the interaction between Spire and Capu in light of the fact that Capu can be regulated by autoinhibition.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to acquire polTIRFM data and then calibrate the setup. Calibration corrects for any systematic variations in beam intensity and unequal detector sensitivities and is performed for each slide after experimental data are recorded. To convert the intensities into angles, one set of (θ, ϕ, δ(s), δ(f), κ) is then determined from one complete cycle of the incident intensities. This process is repeated for every cycle in the trace to measure the time dependence of rotational motions. The collection and analysis of data is similar for the processive motility assay for myosin V and for the twirling filament assay, in which a sparsely labeled actin filament is translocated by a field of unlabeled myosin V.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the processive motility assay for investigating the motility of myosin V in vitro. Biotin-Alexa actin filaments are fixed to a slide by biotin/streptavidin linkages and aligned with the microscope x-axis by fluid flow. The orientation of a rhodamine-calmodulin (CaM) probe bound to a single myosin V molecule is determined as it moves along an actin filament. Excess wild-type calmodulin (WT-CaM) is present in the buffer solution to replenish lost CaM from the myosin lever arm. The techniques for myosin V should be generally applicable to other single-molecule experiments where angular changes have an important mechanistic role in their biological function.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the twirling filament assay, so named because actin sometimes twirls about its own axis as it is translocated by myosin. A gliding filament assay is constructed in which a sparsely labeled actin filament (0.3% of the actin monomers contain 6'- iodoacetamidotetramethylrhodamine [IATR]) is translocated by a field of unlabeled myosin V fixed to the surface. The polTIRFM twirling assay differs from a standard gliding filament assay in that full filaments are not visible, but rather individual fluorophores are spaced along each filament. The goal is to investigate possible rotational motions of the actin filament about its axis (i.e., twirling) by measuring the spatial angle of the fluorescent probe as a function of time. Successful assays contain microscopic fields of approximately 50 isolated points of fluorescence that move across the field in the presence of ATP. Actin is usually translocated by more than one myosin molecule, depending on the filament length and the myosin surface density. Sparsely labeled filaments are required because the orientation of only one probe can be resolved at a time.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to construct sample chambers (flow chambers) for polTIRFM motility assays. Each chamber can hold ∼20 µL of solution. To flow a solution through the chamber, the solution is added to the chamber with a pipette while wicking out the previous contents with filter paper. Each end of the coverslip should extend beyond the edge of the slide to support the pipette tip and filter paper. The flow rate can be roughly controlled by adjusting the contact area between the filter paper and the solution. The chambers can be used for investigating the motility of myosin V in vitro with the processive motility assay, as well as for assessing the motility of actin using the twirling assay.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (∼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to label chicken calmodulin (CaM) with bifunctional rhodamine (BR) at two engineered cysteine (Cys) residues (P66C and A73C) so that it cross-links the two Cys sites. The resulting BR-CaM protein is then purified by high-performance liquid chromatography (HPLC) and concentrated by filter centrifugation. To confirm that the two Cys residues in the labeled CaM are actually cross-linked by BR, a sample of purified BR-CaM is digested by an endoproteinase and analyzed by mass spectrometry. The BR-CaM can then be used to label myosin V, which can in turn be used in a polTIRFM processive motility assay.

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.

Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila melanogaster oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. We find that the interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming.

A new approach is presented for measuring the three-dimensional orientation of individual macromolecules using single molecule fluorescence polarization (SMFP) microscopy. The technique uses the unique polarizations of evanescent waves generated by total internal reflection to excite the dipole moment of individual fluorophores. To evaluate the new SMFP technique, single molecule orientation measurements from sparsely labeled F-actin are compared to ensemble-averaged orientation data from similarly prepared densely labeled F-actin. Standard deviations of the SMFP measurements taken at 40 ms time intervals indicate that the uncertainty for individual measurements of axial and azimuthal angles is approximately 10 degrees at 40 ms time resolution. Comparison with ensemble data shows there are no substantial systematic errors associated with the single molecule measurements. In addition to evaluating the technique, the data also provide a new measurement of the torsional rigidity of F-actin. These measurements support the smaller of two values of the torsional rigidity of F-actin previously reported.

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <<10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.

Several complementary techniques have been developed to determine average orientation, dynamics on multiple time scales, and concerted rotational motions of individual fluorescent probes bound to biological macromolecules. In both protein domains and nucleic acids, tilting and wobble are relevant to their functional mechanisms. Here we briefly review methods to detect angles and rotational motions of single fluorophores and give an example of three-dimensional, total internal reflection, single-molecule fluorescence polarization applied to actin as it is translocated by conventional muscle myosin.

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.