Nuclear actin dynamics and functions at a glance.

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development.

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation.

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Insights into the Mechanism of Action of the Degraded Limonoid Prieurianin.

Limonoids are extremely diversified in plants, with many categories of products bearing an intact, rearranged or fragmented oxygenated scaffold. A specific subgroup of fragmented or degraded limonoids derives from the tetranortriterpenoid prieurianin, initially isolated from the tree Trichilia prieuriana but also found in other plants of the Meliaceae family, including the more abundant species Aphanamixis polystachya. Prieurianin-type limonoids include about seventy compounds, among which are dregeanin and rohitukin. Prieurianin and analogs exhibit insecticidal, antimicrobial, antiadipogenic and/or antiparasitic properties but their mechanism of action remains ill-defined at present. Previous studies have shown that prieurianin, initially known as endosidin 1, stabilizes the actin cytoskeleton in plant and mammalian cells via the modulation of the architecture and dynamic of the actin network, most likely via interference with actin-binding proteins. A new mechanistic hypothesis is advanced here based on the recent discovery of the targeting of the chaperone protein Hsp47 by the fragmented limonoid fraxinellone. Molecular modeling suggested that prieurianin and, to a lesser extent dregeanin, can form very stable complexes with Hsp47 at the protein-collagen interface. Hsp-binding may account for the insecticidal action of the product. The present review draws up a new mechanistic portrait of prieurianin and provides an overview of the pharmacological properties of this atypical limonoid and its chemical family.

Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

Zyxin is important for the stability and function of podocytes, especially during mechanical stretch.

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch.

Myosin's powerstroke transitions define atomic scale movement of cardiac thin filament tropomyosin.

Dynamic interactions between the myosin motor head on thick filaments and the actin molecular track on thin filaments drive the myosin-crossbridge cycle that powers muscle contraction. The process is initiated by Ca2+ and the opening of troponin-tropomyosin-blocked myosin-binding sites on actin. The ensuing recruitment of myosin heads and their transformation from pre-powerstroke to post-powerstroke conformation on actin produce the force required for contraction. Cryo-EM-based atomic models confirm that during this process, tropomyosin occupies three different average positions on actin. Tropomyosin pivoting on actin away from a TnI-imposed myosin-blocking position accounts for part of the Ca2+ activation observed. However, the structure of tropomyosin on thin filaments that follows pre-powerstroke myosin binding and its translocation during myosin's pre-powerstroke to post-powerstroke transition remains unresolved. Here, we approach this transition computationally in silico. We used the myosin helix-loop-helix motif as an anchor to dock models of pre-powerstroke cardiac myosin to the cleft between neighboring actin subunits along cardiac thin filaments. We then performed targeted molecular dynamics simulations of the transition between pre- and post-powerstroke conformations on actin in the presence of cardiac troponin-tropomyosin. These simulations show Arg 369 and Glu 370 on the tip of myosin Loop-4 encountering identically charged residues on tropomyosin. The charge repulsion between residues causes tropomyosin translocation across actin, thus accounting for the final regulatory step in the activation of the thin filament, and, in turn, facilitating myosin movement along the filament. We suggest that during muscle activity, myosin-induced tropomyosin movement is likely to result in unencumbered myosin head interactions on actin at low-energy cost.

Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

BioID Analysis of Actin-Binding Proteins.

Proteins often exist and function as part of higher-order complexes or networks. A challenge is to identify the universe of proximal and interacting partners for a given protein. We describe how the high-activity promiscuous biotin ligase called TurboID is fused to the actin-binding peptide LifeAct to label by biotinylation proteins that bind, or are in close proximity, to actin. The rapid enzyme kinetics of TurboID allows the profiles of actin-binding proteins to be compared under different conditions, such as acute disruption of filamentous actin structures with cytochalasin D.

[Advances in the study of the actin nucleation factor Spire].

There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

F-actin architecture determines the conversion of chemical energy into mechanical work.

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Myosin XI, a model of its conserved role in plant cell tip growth.

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.

Dynamic interplay of microtubule and actomyosin forces drive tissue extension.

In order to shape a tissue, individual cell-based mechanical forces have to be integrated into a global force pattern. Over the last decades, the importance of actomyosin contractile arrays, which are the key constituents of various morphogenetic processes, has been established for many tissues. Recent studies have demonstrated that the microtubule cytoskeleton mediates folding and elongation of the epithelial sheet during Drosophila morphogenesis, placing microtubule mechanics on par with actin-based processes. While these studies establish the importance of both cytoskeletal systems during cell and tissue rearrangements, a mechanistic understanding of their functional hierarchy is currently missing. Here, we dissect the individual roles of these two key generators of mechanical forces during epithelium elongation in the developing Drosophila wing. We show that wing extension, which entails columnar-to-cuboidal cell shape remodeling in a cell-autonomous manner, is driven by anisotropic cell expansion caused by the remodeling of the microtubule cytoskeleton from apico-basal to planarly polarized. Importantly, cell and tissue elongation is not associated with Myosin activity. Instead, Myosin II exhibits a homeostatic role, as actomyosin contraction balances polarized microtubule-based forces to determine the final cell shape. Using a reductionist model, we confirm that pairing microtubule and actomyosin-based forces is sufficient to recapitulate cell elongation and the final cell shape. These results support a hierarchical mechanism whereby microtubule-based forces in some epithelial systems prime actomyosin-generated forces.

Variance and higher moments in the sigmoidal self-assembly of branched fibrils.

Self-assembly of functional branched filaments, such as actin filaments and microtubules, or dysfunctional ones, such as amyloid fibrils, plays important roles in many biological processes. Here, based on the master equation approach, we study the kinetics of the formation of the branched fibrils. In our model, a branched fibril has one mother branch and several daughter branches. A daughter branch grows from the side of a pre-existing mother branch or daughter branch. In our model, we consider five basic processes for the self-assembly of the branched filaments, namely, the nucleation, the dissociation of the primary nucleus of fibrils, the elongation, the fragmentation, and the branching. The elongation of a mother branch from two ends and the elongation of a daughter branch from two ends can, in principle, occur with four different rate constants associated with the corresponding tips. This leads to a pronounced impact of the directionality of growth on the kinetics of the self-assembly. Here, we have unified and generalized our four previously presented models of branched fibrillogenesis in a single model. We have obtained a system of non-linear ordinary differential equations that give the time evolution of the polymer numbers and the mass concentrations along with the higher moments as observable quantities.

Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex.

Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeFx bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeFx transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of Pi from the ADP-Pi transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.

Regulation of Epithelial and Endothelial Barriers by Molecular Chaperones.

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin ß4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells.

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.