Mechanisms of actin filament severing and elongation by formins.

Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3-7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8-11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.

The nematode α-catenin ortholog, HMP1, has an extended α-helix when bound to actin filaments.

The regulation of cell-cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to filamentous actin at cell-cell junctions. The cadherin-catenin complex plays key roles in cell physiology, organism development, and disease. While mutagenesis of Caenorhabditis elegans cadherin and catenin shows that these proteins are key for embryonic morphogenesis, we know surprisingly little about their structure and attachment to the cytoskeleton. In contrast to mammalian α-catenin that functions as a dimer or monomer, the α-catenin ortholog from C. elegans, HMP1 for humpback, is a monomer. Our cryogenic electron microscopy (cryoEM) structure of HMP1/α-catenin reveals that the amino- and carboxy-terminal domains of HMP1/α-catenin are disordered and not in contact with the remaining HMP1/α-catenin middle domain. Since the carboxy-terminal HMP1/α-catenin domain is the F-actin-binding domain (FABD), this interdomain constellation suggests that HMP1/α-catenin is constitutively active, which we confirm biochemically. Our perhaps most surprising finding, given the high sequence similarity between the mammalian and nematode proteins, is our cryoEM structure of HMP1/α-catenin bound to F-actin. Unlike the structure of mammalian α-catenin bound to F-actin, binding to F-actin seems to allosterically convert a loop region of the HMP1/α-catenin FABD to extend an HMP1/α-catenin FABD α-helix. We use cryoEM and bundling assays to show for the first time how the FABD of HMP1/α-catenin bundles actin in the absence of force. Collectively, our data advance our understanding of α-catenin regulation of cell-cell contacts and additionally aid our understanding of the evolution of multicellularity in metazoans.

Bending forces and nucleotide state jointly regulate F-actin structure.

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation1-3. In turn, force4,5 and actin filament (F-actin) nucleotide state6 regulate actin dynamics by tuning F-actin's engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP-F-actin and ADP-Pi-F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP-F-actin and ADP-Pi-F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.

Structural basis of actin filament assembly and aging.

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state1-3. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+ at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca2+-actin shows slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

Molecular Tuning of Actin Dynamics in Leukocyte Migration as Revealed by Immune-Related Actinopathies.

Motility is a crucial activity of immune cells allowing them to patrol tissues as they differentiate, sample or exchange information, and execute their effector functions. Although all immune cells are highly migratory, each subset is endowed with very distinct motility patterns in accordance with functional specification. Furthermore individual immune cell subsets adapt their motility behaviour to the surrounding tissue environment. This review focuses on how the generation and adaptation of diversified motility patterns in immune cells is sustained by actin cytoskeleton dynamics. In particular, we review the knowledge gained through the study of inborn errors of immunity (IEI) related to actin defects. Such pathologies are unique models that help us to uncover the contribution of individual actin regulators to the migration of immune cells in the context of their development and function.

Ultrastructural analysis in yeast reveals a meiosis-specific actin-containing nuclear bundle.

Actin polymerises to form filaments/cables for motility, transport, and the structural framework in a cell. Recent studies show that actin polymers are present not only in the cytoplasm but also in the nuclei of vertebrate cells. Here, we show, by electron microscopic observation with rapid freezing and high-pressure freezing, a unique bundled structure containing actin in the nuclei of budding yeast cells undergoing meiosis. The nuclear bundle during meiosis consists of multiple filaments with a rectangular lattice arrangement, often showing a feather-like appearance. The bundle was immunolabelled with an anti-actin antibody and was sensitive to an actin-depolymerising drug. Similar to cytoplasmic bundles, nuclear bundles are rarely seen in premeiotic cells and spores and are induced during meiotic prophase-I. The formation of the nuclear bundle is independent of DNA double-stranded breaks. We speculate that nuclear bundles containing actin play a role in nuclear events during meiotic prophase I.

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure.

Atomic force microscopy (AFM) can visualize functional biomolecules near the physiological condition, but the observed data are limited to the surface height of specimens. Since the AFM images highly depend on the probe tip shape, for successful inference of molecular structures from the measurement, the knowledge of the probe shape is required, but is often missing. Here, we developed a method of the rigid-body fitting to AFM images, which simultaneously finds the shape of the probe tip and the placement of the molecular structure via an exhaustive search. First, we examined four similarity scores via twin-experiments for four test proteins, finding that the cosine similarity score generally worked best, whereas the pixel-RMSD and the correlation coefficient were also useful. We then applied the method to two experimental high-speed-AFM images inferring the probe shape and the molecular placement. The results suggest that the appropriate similarity score can differ between target systems. For an actin filament image, the cosine similarity apparently worked best. For an image of the flagellar protein FlhAC, we found the correlation coefficient gave better results. This difference may partly be attributed to the flexibility in the target molecule, ignored in the rigid-body fitting. The inferred tip shape and placement results can be further refined by other methods, such as the flexible fitting molecular dynamics simulations. The developed software is publicly available.

Super-resolved visualization of single DNA-based tension sensors in cell adhesion.

Cell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20-30 nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding.

Histologic, immunohistochemical, and scanning electron microscopic comparison of pre-iridal monocellular and fibrovascular membranes in normal and glaucomatous canine globes.

OBJECTIVES: (i) To evaluate immunohistochemical labeling of pre-iridal monocellular and fibrovascular membranes and (ii) describe the light and scanning electron microscopic (SEM) characteristics of these membranes in glaucomatous and normal/control canine globes. MATERIALS AND METHODS: All globes were evaluated with light microscopy. Immunohistochemical labeling for CD18, Smooth muscle actin (SMA), and CD117 was completed on 40 canine globes with congenital/anterior segment dysgenesis-associated glaucoma (n = 10), primary/goniodysgenesis-associated glaucoma (n = 10), secondary glaucoma (n = 10), and normal/control globes (n = 10). SEM was completed on 10 globes: 5 with monocellular membranes, 3 with fibrovascular membranes, and 2 without a histologically detectable membrane. RESULTS: Monocellular membranes were detected in all normal/control globes with light microscopy and appeared to be morphologically very similar to those in diseased globes. CD18 labeling was detected in 9/10 monocellular membranes in normal/control globes, 15/23 monocellular, and 7/8 fibrovascular membranes in globes with glaucoma. SMA and CD117 labeling was not detected in monocellular membranes of normal/control globes. SMA was expressed in 10/23 monocellular and 7/8 fibrovascular membranes of glaucomatous globes. CD117 was expressed in 7/23 monocellular and 5/8 fibrovascular membranes of glaucomatous globes. SEM of monocellular membranes revealed a continuous sheet of mostly spindle cells and few individual round cells that extended over the anterior iris face in normal/control and all glaucomatous globes. CONCLUSION: Pre-iridal monocellular membranes are a normal component of the anterior iris surface, and CD18 immunoreactivity suggests some cells within these are of leukocytic origin. SMA and CD117 labeling of monocellular membranes in glaucomatous, but not normal/control globes, suggest metaplastic cellular change secondary to intraocular pathology related to glaucoma.

A mechanogenetic role for the actomyosin complex in branching morphogenesis of epithelial organs.

The actomyosin complex plays crucial roles in various life processes by balancing the forces generated by cellular components. In addition to its physical function, the actomyosin complex participates in mechanotransduction. However, the exact role of actomyosin contractility in force transmission and the related transcriptional changes during morphogenesis are not fully understood. Here, we report a mechanogenetic role of the actomyosin complex in branching morphogenesis using an organotypic culture system of mouse embryonic submandibular glands. We dissected the physical factors arranged by characteristic actin structures in developing epithelial buds and identified the spatial distribution of forces that is essential for buckling mechanism to promote the branching process. Moreover, the crucial genes required for the distribution of epithelial progenitor cells were regulated by YAP and TAZ through a mechanotransduction process in epithelial organs. These findings are important for our understanding of the physical processes involved in the development of epithelial organs and provide a theoretical background for developing new approaches for organ regeneration.

Clusters of a Few Bound Cofilins Sever Actin Filaments.

Cofilin is an essential actin filament severing protein that accelerates the assembly dynamics and turnover of actin networks by increasing the number of filament ends where subunits add and dissociate. It binds filament subunits stoichiometrically and cooperatively, forming clusters of contiguously-bound cofilin at sub-saturating occupancies. Filaments partially occupied with cofilin sever at boundaries between bare and cofilin-decorated segments. Imaging studies concluded that bound clusters must reach a critical size (Cc) of 13-100 cofilins to sever filaments. In contrast, structural and modeling studies suggest that a few or even a single cofilin can sever filaments, possibly with different severing rate constants. How clusters grow through the cooperative incorporation of additional cofilin molecules, specifically if they elongate asymmetrically or uniformly from both ends and if they are modulated by filament shape and external force, also lacks consensus. Here, using hydrodynamic flow to visualize individual actin filaments with TIRF microscopy, we found that neither flow-induced filament bending, tension, nor surface attachment conditions substantially affected the kinetics of cofilin binding to actin filaments. Clusters of bound cofilin preferentially extended toward filament pointed ends and displayed severing competency at small sizes (Cc < 3), with no detectable severing dependence on cluster size. These data support models in which small clusters of cofilin introduce local, but asymmetric, structural changes in actin filaments that promote filament severing with a rate constant that depends weakly on the size of the cluster.

Ultrastructural clarification of the peripherally located actin network in the myelinated axons / Aclaración ultraestructural de la red de actina localizada periféricamente en los axones mielinizados

SUMMARY: Despite the existence of a large amount of actin in the axons, the concentration F-actin was quite low in the myelinated axons and almost all the F-actin were located in the peripheries of the myelinated axons. Until now, the ultrastructural localization of F-actin has still not been reported in the myelinated axons, probably due to the lack of an appropriate detection method. In the present study, a phalloidin-based FITC-anti-FITC technique was adopted to investigate the subcellular localization of F-actin in the myelinated axons. By using this technique, F-actin is located in the outer and inner collars of myelinated cytoplasm surrounding the intermodal axon, the Schmidt-Lanterman incisures, the paranodal terminal loops and the nodal microvilli. In addition, the satellite cell envelope, which encapsulates the axonal initial segment of the peripheral sensory neuron, was also demonstrated as an F-actin-enriched structure. This study provided a hitherto unreported ultrastructural view of the F-actin in the myelinated axons, which may assist in understanding the unique organization of axonal actin cytoskeleton.

RESUMEN: A pesar de la existencia de una gran cantidad de actina en los axones, la concentración de F-actina era bastante baja en los axones mielinizados y casi la totalidad de F-actina se localizaba en las periferias de los axones mielinizados. A la fecha aún no se ha reportado la localización ultraestructural de F-actina en los axones mielinizados, probablemente debido a la falta de un método de detección apropiado. En el presente estudio, se adoptó una técnica FITC-anti-FITC basada en faloidina para investigar la localización subcelular de F-actina en los axones mielinizados. Mediante el uso de esta técnica, la F-actina se localiza en los collares externo e interno del citoplasma mielinizado que rodea el axón intermodal, a las incisiones de Schmidt-Lanterman,a las asas terminales paranodales y a las microvellosidades nodales. Además, la envoltura de la célula satélite, que encapsula el segmento axonal inicial de la neurona sensorial periférica, también se demostró como una estructura enriquecida con F-actina. Este estudio proporcionó una vista ultraestructural de la F-actina en los axones mielinizados, que puede ayudar a comprender la organización única del citoesqueleto de actina axonal.

Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy.

Synthesis and multiple STED imaging applications of four, red-emitting (610-670 nm), tetrazine-functionalized fluorescent probes (CBRD = Chemical Biology Research group Dye 1-4) with large Stokes-shift is presented. Present studies revealed the super-resolution microscopy applicability of the probes as demonstrated through bioorthogonal labeling scheme of cytoskeletal proteins actin and keratin-19, and mitochondrial protein TOMM20. Furthermore, super-resolved images of insulin receptors in live-cell bioorthogonal labeling schemes through a genetically encoded cyclooctynylated non-canonical amino acid are also presented. The large Stokes-shifts and the wide spectral bands of the probes enabled the use of two common depletion lasers (660 nm and 775 nm). The probes were also found suitable for super-resolution microscopy in combination with two-photon excitation (2P-STED) resulting in improved spatial resolution. One of the dyes was also used together with two commercial dyes in the three-color STED imaging of intracellular structures.

Stress fibres are embedded in a contractile cortical network.

Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modelling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of Oryctolagus cuniculus.

To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.

Structure of the Lifeact-F-actin complex.

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs.

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Pseudo-obstruction-inducing ACTG2R257C alters actin organization and function.

Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.

Recombinant Human Soluble Thrombomodulin Suppresses Monocyte Adhesion by Reducing Lipopolysaccharide-Induced Endothelial Cellular Stiffening.

Endothelial cellular stiffening has been observed not only in inflamed cultured endothelial cells but also in the endothelium of atherosclerotic regions, which is an underlying cause of monocyte adhesion and accumulation. Although recombinant soluble thrombomodulin (rsTM) has been reported to suppress the inflammatory response of endothelial cells, its role in regulating endothelial cellular stiffness remains unclear. The purpose of this study was to investigate the impact of anticoagulant rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening. We show that LPS increases endothelial cellular stiffness by using atomic force microscopy and that rsTM reduces LPS-induced cellular stiffening not only through the attenuation of actin fiber and focal adhesion formation but also via the improvement of gap junction functionality. Moreover, post-administration of rsTM, after LPS stimulation, attenuated LPS-induced cellular stiffening. We also found that endothelial cells regulate leukocyte adhesion in a substrate- and cellular stiffness-dependent manner. Our result show that LPS-induced cellular stiffening enhances monocytic THP-1 cell line adhesion, whereas rsTM suppresses THP-1 cell adhesion to inflamed endothelial cells by reducing cellular stiffness. Endothelial cells increase cellular stiffness in reaction to inflammation, thereby promoting monocyte adhesion. Treatment of rsTM reduced LPS-induced cellular stiffening and suppressed monocyte adhesion in a cellular stiffness-dependent manner.