A sustainable nanocellulose-based superabsorbent from kapok fiber with advanced oil absorption and recyclability.

Creating a low-cost, highly efficient, and recyclable superabsorbent for spilled-oil cleanup is of great significance but remains a big challenge. Herein, we report a facile strategy to produce economic, environmentally friendly, and reusable foam from agricultural waste kapok fibers. These kapok-derived cellulose nanofibrils foams (KNFs) demonstrate a hierarchically porous structure at micro-level with ultra-low density (2.7 mg·cm-3). The superhydrophobic KNFs (150.5°) show outstanding oil absorption (126.8-320.4 g·g-1) and oil-water separation performance. Notably, a facile approach is designed to reuse KNFs easily by a homemade oil release system. The release behavior of the KNFs is quantitatively analyzed and confirmed by the Rigter-Peppas model, indicating that the oil release followed the Fickian diffusion. The KNFs exhibit desirable reusability, and can be recycled for at least 50 times while keeping excellent oil absorption, and release performance. These advantages prove that the KNF is a desirable substitute for spilled-oil treatment.

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism.

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening.

Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.

Mechanical and migratory properties of normal, scar, and Dupuytren's fibroblasts.

Mechanical properties of myofibroblasts play a key role in Dupuytren's disease. Here, we used atomic force microscopy to measure the viscoelastic properties of 3 different types of human primary fibroblasts derived from a same patient: normal and scar dermal fibroblasts and palmar fascial fibroblasts from Dupuytren's nodules. Different stiffness hydrogels (soft ~1 kPa and stiff ~ 50 kPa) were used as cell culture matrix to mimic the mechanical properties of the natural tissues, and atomic force microscopy step response force curves were used to discriminate between elastic and viscous properties of cells. Since transforming growth factor-ß1 (TGF-ß1) is known to induce expression of α-smooth muscle actin positive stress fibers in myofibroblasts, we investigated the behavior of these fibroblasts before and after applying TGF-ß1. Finally, we performed an in vitro cell motility test, the wound healing or scratch assay, to evaluate the migratory properties of these fibroblasts. We found that (1) Dupuytren's fibroblasts are stiffer than normal and scar fibroblasts, the elastic modulus E ranging from 4.4, 2.1, to 1.8 kPa, for Dupuytren's, normal and scar fibroblasts, respectively; (2) TGF-ß1 enhances the level of α-smooth muscle actin expression and thus cell stiffness in Dupuytren's fibroblasts (E, ~6.2 kPa); (3) matrix stiffness influences cell mechanical properties most prominently in Dupuytren's fibroblasts; and (4) Dupuytren's fibroblasts migrate slower than the other fibroblasts by a factor of 3. Taking together, our results showed that mechanical and migratory properties of fibroblasts might help to discriminate between different pathological conditions, helping to identify and recognize specific cell phenotypes.

Actin Filament Structures in Migrating Cells.

Cell migration is necessary for several developmental processes in multicellular organisms. Furthermore, many physiological processes such as wound healing and immunological events in adult animals are dependent on cell migration. Consequently, defects in cell migration are linked to various diseases including immunological disorders as well as cancer progression and metastasis formation. Cell migration is driven by specific protrusive and contractile actin filament structures, but the types and relative contributions of these actin filament arrays vary depending on the cell type and the environment of the cell. In this chapter, we introduce the most important actin filament structures that contribute to mesenchymal and amoeboid cell migration modes and discuss the mechanisms by which the assembly and turnover of these structures are controlled by various actin-binding proteins.

How actin/myosin crosstalks guide the adhesion, locomotion and polarization of cells.

Cell-tissue-tissue interaction is determined by specific short range forces between cell adhesion molecules (CAMs) and ligands of the tissue, long range repulsion forces mediated by cell surface grafted macromolecules and adhesion-induced elastic stresses in the cell envelope. This interplay of forces triggers the rapid random clustering of tightly coupled linkers. By coupling of actin gel patches to the intracellular domains of the CAMs, these clusters can grow in a secondary process resulting in the formation of functional adhesion microdomains (ADs). The ADs can act as biochemical steering centers by recruiting and activating functional proteins, such as GTPases and associated regulating proteins, through electrostatic-hydrophobic forces with cationic lipid domains that act as attractive centers. First, I summarize physical concepts of cell adhesion revealed by studies of biomimetic systems. Then I describe the role of the adhesion domains as biochemical signaling platforms and force transmission centers promoting cellular protrusions, in terms of a shell string model of cells. Protrusion forces are generated by actin gelation triggered by molecular machines (focal adhesion kinase (FAK), Src-kinases and associated adaptors) which assemble around newly formed integrin clusters. They recruit and activate the GTPases Rac-1 and actin gelation promoters to charged membrane domains via electrostatic-hydrophobic forces. The cell front is pushed forward in a cyclic and stepwise manner and the step-width is determined by the dynamics antagonistic interplay between Rac-1 and RhoA. The global cell polarization in the direction of motion is mediated by the actin-microtubule (MT) crosstalk at adhesion domains. Supramolecular actin-MT assemblies at the front help to promote actin polymerization. At the rear they regulate the dismantling of the ADs through the Ca(++)-mediated activation of the protease calpain and trigger their disruption by RhoA mediated contraction via stress fibers. This article is part of a Special Issue entitled: Mechanobiology.

Cell shape dynamics reveal balance of elasticity and contractility in peripheral arcs.

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension.

Model-based traction force microscopy reveals differential tension in cellular actin bundles.

Adherent cells use forces at the cell-substrate interface to sense and respond to the physical properties of their environment. These cell forces can be measured with traction force microscopy which inverts the equations of elasticity theory to calculate them from the deformations of soft polymer substrates. We introduce a new type of traction force microscopy that in contrast to traditional methods uses additional image data for cytoskeleton and adhesion structures and a biophysical model to improve the robustness of the inverse procedure and abolishes the need for regularization. We use this method to demonstrate that ventral stress fibers of U2OS-cells are typically under higher mechanical tension than dorsal stress fibers or transverse arcs.

Vinculin tension distributions of individual stress fibers within cell-matrix adhesions.

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

Hyperthermia induces cytoskeletal alterations and mitotic catastrophe in p53-deficient H1299 lung cancer cells.

Hyperthermia is used in cancer therapy, however much remains to be discovered regarding its mechanisms of action at the cellular level. In this study, the effects of hyperthermia on cell death, survival, morphology and the cytoskeleton were investigated in a non-small cell lung cancer cell line, H1299. Despite the fact that this cell line is widely used in research, it has not yet been tested for heat shock sensitivity. Cells were given a 30-min heat shock at 43.5°C and 45°C and left to recover at 37°C for 24 and 48 h. 24 h after heat shock treatment, we monitored changes in the organization of the cytoskeleton using immunofluorescence microscopy. The number of actin stress fibers was significantly reduced, microtubules formed a looser meshwork, a portion of the cells possessed multipolar mitotic spindles, whereas vimentin filaments collapsed into perinuclear complexes. 48 h following heat stress, most of the cells showed recovery of the cytoskeleton, however we observed a considerable number of giant cells that were multinucleated or contained one enlarged nucleus. The data obtained by MTT assay showed a dose-dependent decrease of cell viability, while flow cytometric analysis revealed an increase in the number of cells with externalized phosphatidylserine. The results suggest that one of the modes of heat-induced cell death in H1299 cells is mitotic catastrophe, which probably ends in apoptosis.

Contraction of cross-linked actomyosin bundles.

Cross-linked actomyosin bundles retract when severed in vivo by laser ablation, or when isolated from the cell and micromanipulated in vitro in the presence of ATP. We identify the timescale for contraction as a viscoelastic time τ, where the viscosity is due to (internal) protein friction. We obtain an estimate of the order of magnitude of the contraction time τ ≈ 10-100 s, consistent with available experimental data for circumferential microfilament bundles and stress fibers. Our results are supported by an exactly solvable, hydrodynamic model of a retracting bundle as a cylinder of isotropic, active matter, from which the order of magnitude of the active stress is estimated.

Simultaneous contraction and buckling of stress fibers in individual cells.

It has been proposed that buckling of actin stress fibers (SFs) may be associated with their disassembly. However, much of the detail remains unknown partly because the use of an elastic membrane sheet, conventionally necessary for inducing SF buckling with a mechanical compression to adherent cells, may limit high quality and quick imaging of the dynamic cellular events. Here, we present an alternate approach to induce buckling behavior of SFs on a readily observable glass plate. Actin SFs were extracted from cells, and constituent myosin II (MII) molecules were partially photo-inactivated in contractility. An addition of Mg-ATP allowed actin-myosin cross-bridge cycling and resultant contraction of only thick SFs that still contained active MII in the large volume. Meanwhile, thin SFs with virtually no active motor protein in the small volume had no choice but to buckle with the shortening movement of nearby thick SFs functioning as a compression-inducing element. This novel technique, thus allowing for selective inductions of contraction and buckling of SFs and measurements of the cellular prestress, may be applicable to not only investigations on their disassembly mechanisms but also to measurements of the relative thickness of individual SFs in each cell.

Viscoelastic response of contractile filament bundles.

The actin cytoskeleton of adherent tissue cells often condenses into filament bundles contracted by myosin motors, so-called stress fibers, which play a crucial role in the mechanical interaction of cells with their environment. Stress fibers are usually attached to their environment at the endpoints, but possibly also along their whole length. We introduce a theoretical model for such contractile filament bundles which combines passive viscoelasticity with active contractility. The model equations are solved analytically for two different types of boundary conditions. A free boundary corresponds to stress fiber contraction dynamics after laser surgery and results in good agreement with experimental data. Imposing cyclic varying boundary forces allows us to calculate the complex modulus of a single stress fiber.

Regulated binding of adenomatous polyposis coli protein to actin.

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. Here we show that purified carboxyl-terminal basic domain of human APC protein (APC-basic) bound directly to and bundled actin filaments and associated with actin stress fibers in microinjected cells. Actin filaments and microtubules competed for binding to APC-basic, but APC-basic also could cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro were inhibited by its ligand EB1, and co-microinjection of EB1 prevented APC association with stress fibers. Point mutations in EB1 that disrupted APC binding relieved the inhibition in vitro and restored APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression.

Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.

Stress fibers play a central role in adhesion, motility, and morphogenesis of eukaryotic cells, but the mechanism of how these and other contractile actomyosin structures are generated is not known. By analyzing stress fiber assembly pathways using live cell microscopy, we revealed that these structures are generated by two distinct mechanisms. Dorsal stress fibers, which are connected to the substrate via a focal adhesion at one end, are assembled through formin (mDia1/DRF1)-driven actin polymerization at focal adhesions. In contrast, transverse arcs, which are not directly anchored to substrate, are generated by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Remarkably, dorsal stress fibers and transverse arcs can be converted to ventral stress fibers anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that the rapid association-dissociation kinetics of cross-linkers may be essential for the formation and contractility of stress fibers. Based on these data, we propose a general model for assembly and maintenance of contractile actin structures in cells.

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells.

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Expression of the actin stress fiber-associated protein CLP36 in the human placenta.

Differentiation processes in the trophoblast comprise polarization, cell fusion and migration. All these processes involve dramatic reorganizations of cytoskeletal proteins such as intermediate filaments or actin. Due to very restricted knowledge on cytoskeletal changes in trophoblast, we analyzed the protein expression of an actin stress fiber-associated protein, the carboxy-terminal LIM domain protein (CLP36). CLP36 belongs to the enigma family of proteins, binds to alpha-actinin and is involved in the cytoskeletal reorganization and signal transduction of a variety of cells. CLP36 protein was found to be exclusively expressed in the cytotrophoblast layer. Colocalization of CLP36 with Mib-1 revealed that CLP36 protein expression is restricted to proliferative and early post-proliferative trophoblast cells. Blockage of syncytial fusion by culture of villous explants in the presence of caspase 8 inhibitors further supported this notion since CLP36 was only found in the basal and proliferative layer of the multilayered cytotrophoblast. We present evidence for the exclusive protein expression of CLP36 in proliferative and early post-proliferative trophoblast cells. Pathological pregnancy syndromes such as preeclampsia are driven by alterations of trophoblast differentiation and turnover, where it needs to be elucidated whether CLP36 is involved in these alterations.

PAK1 negatively regulates the activity of the Rho exchange factor NET1.

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.

Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin.

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae.