[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.

Medioapical contractile pulses coordinated between cells regulate Drosophila eye morphogenesis.

Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.

Dark-induced chloroplast relocation depends on actin filaments in the liverwort Apopellia endiviifolia along with the light- and cold-induced relocations.

Chloroplasts move to the periclinal walls of cells under weak light to harness light energy for photosynthesis and to anticlinal walls to avoid strong light. These responses involve the cytoskeleton components microtubules and/or actin filaments. In the dark, chloroplasts move to the anticlinal cell walls bordering neighbouring cells (dark-positioning response), but this response in various plants normally requires a prolonged dark incubation period, which has hampered analysis. However, we recently demonstrated the dark-positioning response that can be induced after a short period of dark incubation in the liverwort Apopellia endiviifolia. Here, we investigated whether the cytoskeleton components function in the dark-positioning response of A. endiviifolia cells. Microtubules and actin filaments were fluorescently visualised in A. endiviifolia cells and were disrupted following treatment with the microtubule and actin filament polymerisation inhibitors. The dark-positioning response was unaffected in the cells with disrupted microtubules. By contrast, the dark-positioning response was inhibited by the disruption of actin filaments. The disruption of actin filaments also restricted chloroplast mobility during light- and cold-dependent chloroplast movements in A. endiviifolia. Therefore, the dark-positioning response of A. endiviifolia depends solely on an actin filament-associated motility mechanism, as do the light- and cold-dependent chloroplast responses.

Cell size and actin architecture determine force generation in optogenetically activated cells.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Application of sequential cyclic compression on cancer cells in a flexible microdevice.

Mechanical forces shape physiological structure and function within cell and tissue microenvironments, during which cells strive to restore their shape or develop an adaptive mechanism to maintain cell integrity depending on strength and type of the mechanical loading. While some cells are shown to experience permanent plastic deformation after a repetitive mechanical tensile loading and unloading, the impact of such repetitive compression on deformation of cells is yet to be understood. As such, the ability to apply cyclic compression is crucial for any experimental setup aimed at the study of mechanical compression taking place in cell and tissue microenvironments. Here, we demonstrate such cyclic compression using a microfluidic compression platform on live cell actin in SKOV-3 ovarian cancer cells. Live imaging of the actin cytoskeleton dynamics of the compressed cells was performed for varying pressures applied sequentially in ascending order during cell compression. Additionally, recovery of the compressed cells was investigated by capturing actin cytoskeleton and nuclei profiles of the cells at zero time and 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. As demonstrated in this work, the observation of morphological, cytoskeletal and nuclear differences in compressed and non-compressed cells, with controlled micro-scale mechanical cell compression and recovery and using live-cell imaging, fluorescent tagging and end point assays, can give insights into the mechanics of cancer cells.

A statistical mechanics model for determining the length distribution of actin filaments under cellular tensional homeostasis.

Tensional homeostasis is a cellular process whereby nonmuscle cells such as fibroblasts keep a constant level of intracellular tension and signaling activities. Cells are allowed thanks to tensional homeostasis to adapt to mechanical stress, but the detailed mechanism remains unclear. Here we address from a theoretical point of view what is required for maintaining cellular tensional homeostasis. A constrained optimization problem is formulated to analytically determine the probability function of the length of individual actin filaments (AFs) responsible for sustaining cellular tension. An objective function composed of two entropic quantities measuring the extent of formation and dispersion of AFs within cells is optimized under two constraint functions dictating a constant amount of actin molecules and tension that are arguably the two most salient features of tensional homeostasis. We then derive a specific probability function of AFs that is qualitatively consistent with previous experimental observations, in which short AF populations preferably appear. Regarding the underlying mechanism, our analyses suggest that the constraint for keeping the constant tension level makes long AF populations smaller in number because long AFs have a higher chance to be involved in bearing larger forces. The specific length distribution of AFs is thus required for achieving the constrained objectives, by which individual cells are endowed with the ability to stably maintain a homeostatic tension throughout the cell, thereby potentially allowing cells to locally detect deviation in the tension, keep resulting biological functions, and hence enable subsequent adaptation to mechanical stress. Although minimal essential factors are included given the actual complexity of cells, our approach would provide a theoretical basis for understanding complicated homeostatic and adaptive behavior of the cell.

Actin-driven Golgi apparatus dispersal during collective migration of epithelial cells.

As a sedentary epithelium turns motile during wound healing, morphogenesis, and metastasis, the Golgi apparatus moves from an apical position, above the nucleus, to a basal position. This apical-to-basal repositioning of Golgi is critical for epithelial cell migration. Yet the molecular mechanism underlying it remains elusive, although microtubules are believed to play a role. Using live-cell and super-resolution imaging, we show that at the onset of collective migration of epithelial cells, Golgi stacks get dispersed to create an unpolarized transitional structure, and surprisingly, this dispersal process depends not on microtubules but on actin cytoskeleton. Golgi-actin interaction involves Arp2/3-driven actin projections emanating from the actin cortex, and a Golgi-localized actin elongation factor, MENA. While in sedentary epithelial cells, actin projections intermittently interact with the apically located Golgi, and the frequency of this event increases before the dispersion of Golgi stacks, at the onset of cell migration. Preventing Golgi-actin interaction with MENA-mutants eliminates Golgi dispersion and reduces the persistence of cell migration. Taken together, we show a process of actin-driven Golgi dispersion that is mechanistically different from the well-known Golgi apparatus fragmentation during mitosis and is essential for collective migration of epithelial cells.

Regulation of the Actin Cytoskeleton-Linked Ca2+ Signaling by Intracellular pH in Fertilized Eggs of Sea Urchin.

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg's receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

Ablation of the P21 Gene of Trypanosoma cruzi Provides Evidence of P21 as a Mediator in the Control of Epimastigote and Intracellular Amastigote Replication.

P21 is an immunomodulatory protein expressed throughout the life cycle of Trypanosoma cruzi, the etiologic agent of Chagas disease. In vitro and in vivo studies have shown that P21 plays an important role in the invasion of mammalian host cells and establishment of infection in a murine model. P21 functions as a signal transducer, triggering intracellular cascades in host cells and resulting in the remodeling of the actin cytoskeleton and parasite internalization. Furthermore, in vivo studies have shown that P21 inhibits angiogenesis, induces inflammation and fibrosis, and regulates intracellular amastigote replication. In this study, we used the CRISPR/Cas9 system for P21 gene knockout and investigated whether the ablation of P21 results in changes in the phenotypes associated with this protein. Ablation of P21 gene resulted in a lower growth rate of epimastigotes and delayed cell cycle progression, accompanied by accumulation of parasites in G1 phase. However, P21 knockout epimastigotes were viable and able to differentiate into metacyclic trypomastigotes, which are infective to mammalian cells. In comparison with wild-type parasites, P21 knockout cells showed a reduced cell invasion rate, demonstrating the role of this protein in host cell invasion. However, there was a higher number of intracellular amastigotes per cell, suggesting that P21 is a negative regulator of amastigote proliferation in mammalian cells. Here, for the first time, we demonstrated the direct correlation between P21 and the replication of intracellular amastigotes, which underlies the chronicity of T. cruzi infection.

Tension-dependent stabilization of E-cadherin limits cell-cell contact expansion in zebrafish germ-layer progenitor cells.

Tension of the actomyosin cell cortex plays a key role in determining cell-cell contact growth and size. The level of cortical tension outside of the cell-cell contact, when pulling at the contact edge, scales with the total size to which a cell-cell contact can grow [J.-L. Maître et al., Science 338, 253-256 (2012)]. Here, we show in zebrafish primary germ-layer progenitor cells that this monotonic relationship only applies to a narrow range of cortical tension increase and that above a critical threshold, contact size inversely scales with cortical tension. This switch from cortical tension increasing to decreasing progenitor cell-cell contact size is caused by cortical tension promoting E-cadherin anchoring to the actomyosin cytoskeleton, thereby increasing clustering and stability of E-cadherin at the contact. After tension-mediated E-cadherin stabilization at the contact exceeds a critical threshold level, the rate by which the contact expands in response to pulling forces from the cortex sharply drops, leading to smaller contacts at physiologically relevant timescales of contact formation. Thus, the activity of cortical tension in expanding cell-cell contact size is limited by tension-stabilizing E-cadherin-actin complexes at the contact.

4polar-STORM polarized super-resolution imaging of actin filament organization in cells.

Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules' orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules' localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.

Microtubule organizing centers regulate spindle positioning in mouse oocytes.

During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known about whether microtubules (MTs) participate in regulating these processes. Using mouse oocytes as a model system, we characterize a subset of MT organizing centers that do not contribute directly to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy, and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a major player in regulating spindle positioning.

Extensile to contractile transition in active microtubule-actin composites generates layered asters with programmable lifetimes.

We study a reconstituted composite system consisting of an active microtubule network interdigitated with a passive network of entangled F-actin filaments. Increasing the concentration of filamentous actin controls the emergent dynamics, inducing a transition from turbulent-like flows to bulk contractions. At intermediate concentrations, where the active stresses change their symmetry from anisotropic extensile to isotropic contracting, the composite separates into layered asters that coexist with the background turbulent fluid. Contracted onion-like asters have a radially extending microtubule-rich cortex that envelops alternating layers of microtubules and F-actin. These self-regulating structures undergo internal reorganization, which appears to minimize the surface area and maintain the ordered layering, even when undergoing aster merging events. Finally, the layered asters are metastable structures. Their lifetime, which ranges from minutes to hours, is encoded in the material properties of the composite. These results challenge the current models of active matter. They demonstrate self-organized dynamical states and patterns evocative of those observed in the cytoskeleton do not require precise biochemical regulation, but can arise from purely mechanical interactions of actively driven filamentous materials.

Single-molecule imaging of IQGAP1 regulating actin filament dynamics.

IQGAP is a conserved family of actin-binding proteins with essential roles in cell motility, cytokinesis, and cell adhesion, yet there remains a limited understanding of how IQGAP proteins directly influence actin filament dynamics. To close this gap, we used single-molecule and single-filament total internal reflection fluorescence microscopy to observe IQGAP regulating actin dynamics in real time. To our knowledge, this is the first study to do so. Our results demonstrate that full-length human IQGAP1 forms dimers that stably bind to actin filament sides and transiently cap barbed ends. These interactions organize filaments into thin bundles, suppress barbed end growth, and inhibit filament disassembly. Surprisingly, each activity depends on distinct combinations of IQGAP1 domains and/or dimerization, suggesting that different mechanisms underlie each functional effect on actin. These observations have important implications for how IQGAP functions as an actin regulator in vivo and how it may be regulated in different biological settings.

Force-dependent activation of actin elongation factor mDia1 protects the cytoskeleton from mechanical damage and promotes stress fiber repair.

Plasticity of cell mechanics underlies a wide range of cell and tissue behaviors allowing cells to migrate through narrow spaces, resist shear forces, and safeguard against mechanical damage. Such plasticity depends on spatiotemporal regulation of the actomyosin cytoskeleton, but mechanisms of adaptive change in cell mechanics remain elusive. Here, we report a mechanism of mechanically activated actin polymerization at focal adhesions (FAs), specifically requiring the actin elongation factor mDia1. By combining live-cell imaging with mathematical modeling, we show that actin polymerization at FAs exhibits pulsatile dynamics where spikes of mDia1 activity are triggered by contractile forces. The suppression of mDia1-mediated actin polymerization increases tension on stress fibers (SFs) leading to an increased frequency of spontaneous SF damage and decreased efficiency of zyxin-mediated SF repair. We conclude that tension-controlled actin polymerization acts as a safety valve dampening excessive tension on the actin cytoskeleton and safeguarding SFs against mechanical damage.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture.

The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division.

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell-stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

Microfluidic investigation of the effect of graphene oxide on mechanical properties of cell and actin cytoskeleton networks: experimental and theoretical approaches.

Biomechanical and morphological analysis of the cells is a novel approach for monitoring the environmental features, drugs, and toxic compounds' effects on cells. Graphene oxide (GO) has a broad range of medical applications such as tissue engineering and drug delivery. However, the effects of GO nanosheets on biological systems have not been completely understood. In this study, we focused on the biophysical characteristics of cells and their changes resulting from the effect of GO nanosheets. The biophysical properties of the cell population were characterized as follows: cell stiffness was calculated by atomic force microscopy, cell motility and invasive properties were characterized in the microfluidic chip in which the cells are able to visualize cell migration at a single-cell level. Intracellular actin was stained to establish a quantitative picture of the intracellular cytoskeleton. In addition, to understand the molecular interaction of GO nanosheets and actin filaments, coarse-grained (CG) molecular dynamics (MD) simulations were carried out. Our results showed that GO nanosheets can reduce cell stiffness in MCF7 cells and MDA-MB-231 cell lines and highly inhibited cell migration (39.2%) in MCF-7 and (38.6%) in MDA-MB-231 cell lines through the GO nanosheets-mediated disruption of the intracellular cytoskeleton. In the presence of GO nanosheets, the cell migration of both cell lines, as well as the cell stiffness, significantly decreased. Moreover, after GO nanosheets treatment, the cell actin network dramatically changed. The experimental and theoretical approaches established a quantitative picture of changes in these networks. Our results showed the reduction of the order parameter in actin filaments was 23% in the MCF7 cell line and 20.4% in the MDA-MB-231 cell line. The theoretical studies also showed that the GO nanosheet-actin filaments have stable interaction during MD simulation. Moreover, the 2D free energy plot indicated the GO nanosheet can induce conformational changes in actin filaments. Our findings showed that the GO nanosheets can increase the distance of actin-actin subunits from 3.22 to 3.5 nm and in addition disrupt native contacts between two subunits which lead to separate actin subunits from each other in actin filaments. In this study, the biomechanical characteristics were used to explain the effect of GO nanosheets on cells which presents a novel view of how GO nanosheets can affect the biological properties of cells without cell death. These findings have the potential to be applied in different biomedical applications.

The initial expansion of the C. elegans syncytial germ line is coupled to incomplete primordial germ cell cytokinesis.

The C. elegans germline is organized as a syncytium in which each germ cell possesses an intercellular bridge that is maintained by a stable actomyosin ring and connected to a common pool of cytoplasm, termed the rachis. How germ cells undergo cytokinesis while maintaining this syncytial architecture is not completely understood. Here, we use live imaging to characterize primordial germ cell (PGC) division in C. elegans first-stage larvae. We show that each PGC possesses a stable intercellular bridge that connects it to a common pool of cytoplasm, which we term the proto-rachis. We further show that the first PGC cytokinesis is incomplete and that the stabilized cytokinetic ring progressively moves towards the proto-rachis and eventually integrates with it. Our results support a model in which the initial expansion of the C. elegans syncytial germline occurs by incomplete cytokinesis, where one daughter germ cell inherits the actomyosin ring that was newly formed by stabilization of the cytokinetic ring, while the other inherits the pre-existing stable actomyosin ring. We propose that such a mechanism of iterative cytokinesis incompletion underpins C. elegans germline expansion and maintenance.