Cadherin puncta are interdigitated dynamic actin protrusions necessary for stable cadherin adhesion.

Cadherins harness the actin cytoskeleton to build cohesive sheets of cells using paradoxically weak bonds, but the molecular mechanisms are poorly understood. In one popular model, actin organizes cadherins into large, micrometer-sized clusters known as puncta. Myosin is thought to pull on these puncta to generate strong adhesion. Here, however, we show that cadherin puncta are actually interdigitated actin microspikes generated by actin polymerization mediated by three factors (Arp2/3, EVL, and CRMP-1). The convoluted membranes in these regions give the impression of cadherin clustering by fluorescence microscopy, but the ratio of cadherin to membrane is constant. Nevertheless, these interlocking fingers of membrane are important for adhesion because perturbing their formation disrupts cell adhesion. In contrast, blocking myosin-dependent contractility does not disrupt either the interdigitated microspikes or lateral membrane adhesion. "Puncta" are zones of strong cell-cell adhesion not due to cadherin clustering but that occur because the interdigitated microspikes expand the surface area available for adhesive bond formation and increase the asperity of the cell surface to promote friction between cells.

Actin protrusions push at apical junctions to maintain E-cadherin adhesion.

Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell-cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

Catastrophic actin filament bursting by cofilin, Aip1, and coronin.

Cofilin is an actin filament severing protein necessary for fast actin turnover dynamics. Coronin and Aip1 promote cofilin-mediated actin filament disassembly, but the mechanism is somewhat controversial. An early model proposed that the combination of cofilin, coronin, and Aip1 disassembled filaments in bursts. A subsequent study only reported severing. Here, we used EM to show that actin filaments convert directly into globular material. A monomer trap assay also shows that the combination of all three factors produces actin monomers faster than any two factors alone. We show that coronin accelerates the release of Pi from actin filaments and promotes highly cooperative cofilin binding to actin to create long stretches of polymer with a hypertwisted morphology. Aip1 attacks these hypertwisted regions along their sides, disintegrating them into monomers or short oligomers. The results are consistent with a catastrophic mode of disassembly, not enhanced severing alone.

The actin filament bundling protein α-actinin-4 actually suppresses actin stress fibers by permitting actin turnover.

Cells organize actin filaments into contractile bundles known as stress fibers that resist mechanical stress, increase cell adhesion, remodel the extracellular matrix, and maintain tissue integrity. α-actinin is an actin filament bundling protein that is thought to be essential for stress fiber formation and stability. However, previous studies have also suggested that α-actinin might disrupt fibers, making the true function of this biomolecule unclear. Here we use fluorescence imaging to show that kidney epithelial cells depleted of α-actinin-4 via shRNA or CRISPR/Cas9, or expressing a disruptive mutant make more massive stress fibers that are less dynamic than those in WT cells, leading to defects in cell motility and wound healing. The increase in stress fiber mass and stability can be explained, in part, by increased loading of the filament component tropomyosin onto stress fibers in the absence of α-actinin, as monitored via immunofluorescence. We show using imaging and cosedimentation that α-actinin and tropomyosin compete for binding to F-actin and that tropomyosin shields actin filaments from cofilin-mediated disassembly in vitro and in cells. Perturbing tropomyosin in cells lacking α-actinin-4 results in a complete loss of stress fibers. Our results with α-actinin-4 on stress fiber organization are the opposite of what might have been predicted from previous in vitro biochemistry and further highlight how the complex interactions of multiple proteins competing for filament binding lead to unexpected functions for actin-binding proteins in cells.

Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly.

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.

Caspase-11 regulates cell migration by promoting Aip1-Cofilin-mediated actin depolymerization.

Coordinated regulation of cell migration, cytokine maturation and apoptosis is critical in inflammatory responses. Caspases, a family of cysteine proteases, are known to regulate cytokine maturation and apoptosis. Here, we show that caspase-11, a mammalian pro-inflammatory caspase, regulates cell migration during inflammation. Caspase-11-deficient lymphocytes exhibit a cell-autonomous migration defect in vitro and in vivo. We demonstrate that caspase-11 interacts physically and functionally with actin interacting protein 1 (Aip1), an activator of cofilin-mediated actin depolymerization. The caspase-recruitment domain (CARD) of caspase-11 interacts with the carboxy-terminal WD40 propeller domain of Aip1 to promote cofilin-mediated actin depolymerization. Cells with Aip1 or caspase-11 deficiency exhibit defects in actin dynamics. Using in vitro actin depolymerization assays, we found that caspase-11 and Aip1 work cooperatively to promote cofilin-mediated actin depolymerization. These data demonstrate a novel cell autonomous caspase-mediated mechanism that regulates actin dynamics and mammalian cell migration distinct from the receptor mediated Rho-Rac-Cdc42 pathway.

Corrigendum: Revisiting bacterial cytolethal distending toxin structure and function.

[This corrects the article DOI: 10.3389/fcimb.2023.1289359.].

Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH.

Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

Revisiting bacterial cytolethal distending toxin structure and function.

Cytolethal distending toxins (CDTs) are intracellular-acting bacterial genotoxins generated by a diverse group of mucocutaneous human pathogens. CDTs must successfully bind to the plasma membrane of host cells in order to exert their modulatory effects. Maximal toxin activity requires all three toxin subunits, CdtA, CdtB, and CdtC, which, based primarily on high-resolution structural data, are believed to preassemble into a tripartite complex necessary for toxin activity. However, biologically active toxin has not been experimentally demonstrated to require assembly of the three subunits into a heterotrimer. Here, we experimentally compared concentration-dependent subunit interactions and toxin cellular activity of the Campylobacter jejuni CDT (Cj-CDT). Co-immunoprecipitation and dialysis retention experiments provided evidence for the presence of heterotrimeric toxin complexes, but only at concentrations of Cj-CdtA, Cj-CdtB, and Cj-CdtC several logs higher than required for Cj-CDT-mediated arrest of the host cell cycle at the G2/M interface, which is triggered by the endonuclease activity associated with the catalytic Cj-CdtB subunit. Microscale thermophoresis confirmed that Cj-CDT subunit interactions occur with low affinity. Collectively, our data suggest that at the lowest concentrations of toxin sufficient for arrest of cell cycle progression, mixtures of Cj-CdtA, Cj-CdtB, and Cj-CdtC consist primarily of non-interacting, subunit monomers. The lack of congruence between toxin tripartite structure and cellular activity suggests that the widely accepted model that CDTs principally intoxicate host cells as preassembled heterotrimeric structures should be revisited.

Facioscapulohumeral muscular dystrophy region gene-1 (FRG-1) is an actin-bundling protein associated with muscle-attachment sites.

In vertebrates, overexpression of facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) recapitulates the pathophysiology exhibited by FSHD patients, although the role of FRG1 in FSHD remains controversial and no precise function for FRG1 has been described in any organism. To gain insight into the function and potential role of FRG1 in FSHD, we analyzed the highly conserved Caenorhabditis elegans ortholog, frg-1. C. elegans body-wall muscles contain two distinct subcellular pools of FRG-1: nuclear FRG-1, concentrated in the nucleoli; and cytoplasmic FRG-1, associated with the Z-disk and costamere-like structures known as dense bodies. Functionally, we demonstrate that FRG-1 is an F-actin-bundling protein, consistent with its localization to dense bodies; this activity is conserved in human FRG1. This is particularly intriguing because it places FRG-1 along side the list of dense-body components whose vertebrate orthologs are involved in the myriad myopathies associated with disrupted costameres and Z-disks. Interestingly, overexpressed FRG-1 preferentially accumulates in the nucleus and, when overexpressed specifically from the frg-1 promoter, disrupts the adult ventral muscle structure and organization. Together, these data further support a role for FRG1 overexpression in FSHD pathophysiology and reveal the previously unsuspected direct involvement of FRG-1 in muscle structure and integrity.

Synaptopodin stress fiber and contractomere at the epithelial junction.

The apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and adhesion. Yet, the structural basis for force generation at the apical junction is not fully understood. Here, we describe two synaptopodin-dependent actomyosin structures that are spatially, temporally, and structurally distinct. The first structure is formed by the retrograde flow of synaptopodin initiated at the apical junction, creating a sarcomeric stress fiber that lies parallel to the apical junction. Contraction of the apical stress fiber is associated with either clustering of membrane components or shortening of junctional length. Upon junction maturation, apical stress fibers are disassembled. In mature epithelial monolayer, a motorized "contractomere" capable of "walking the junction" is formed at the junctional vertex. Actomyosin activities at the contractomere produce a compressive force evident by actin filament buckling and measurement with a new α-actinin-4 force sensor. The motility of contractomeres can adjust junctional length and change cell packing geometry during cell extrusion and intercellular movement. We propose a model of epithelial homeostasis that utilizes contractomere motility to support junction rearrangement while preserving the permeability barrier.

Rapid actin monomer-insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1.

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others.

Dynamic stabilization of actin filaments.

We report here that actin filaments in vitro exist in two populations with significantly different shrinkage rates. Newly polymerized filaments shrink rapidly, primarily from barbed ends, at 1.8/s, but as they age they switch to a stable state that shrinks slowly, primarily from pointed ends, at approximately 0.1/s. This dynamic filament stabilization runs opposite to the classical prediction that actin filaments become more unstable with age as they hydrolyze their bound ATP and release phosphate. Upon cofilin treatment, aged filaments revert to a dynamic state that shows accelerated shrinkage from both ends at a combined rate of 5.9/s. In light of recent electron microscopy studies [Orlova A, et al. (2004) Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization. Proc Natl Acad Sci USA 101:17664-17668], we propose that dynamic stabilization arises from rearrangement of the filament structure from a relatively disordered state immediately after polymerization to the canonical Holmes helix, a change that is reversed by cofilin binding. Our results suggest that plasticity in the internal structure of the actin filament may play a fundamental role in regulating actin dynamics and may help cells build actin assemblies with vastly different turnover rates.

Quantitative analysis of actin turnover in Listeria comet tails: evidence for catastrophic filament turnover.

Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.

CD2AP links actin to PI3 kinase activity to extend epithelial cell height and constrain cell area.

Maintaining the correct ratio of apical, basal, and lateral membrane domains is important for epithelial physiology. Here, we show that CD2AP is a critical determinant of epithelial membrane proportions. Depletion of CD2AP or phosphoinositide 3-kinase (PI3K) inhibition results in loss of F-actin and expansion of apical-basal domains, which comes at the expense of lateral membrane height in MDCK cells. We demonstrate that the SH3 domains of CD2AP bind to PI3K and are necessary for PI3K activity along lateral membranes and constraining cell area. Tethering the SH3 domains of CD2AP or p110γ to the membrane is sufficient to rescue CD2AP-knockdown phenotypes. CD2AP and PI3K are both upstream and downstream of actin polymerization. Since CD2AP binds to both actin filaments and PI3K, CD2AP might bridge actin assembly to PI3K activation to form a positive feedback loop to support lateral membrane extension. Our results provide insight into the squamous to cuboidal to columnar epithelial transitions seen in complex epithelial tissues in vivo.

Mechanism of Long-Range Chromosome Motion Triggered by Gene Activation.

Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.

Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex.

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.

A quantitative analysis of contractility in active cytoskeletal protein networks.

Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and alpha-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of approximately 1 microN, or approximately 100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity.

Cell-free extract systems and the cytoskeleton: preparation of biochemical experiments for transmission electron microscopy.

Cell-free systems can be used to reconstitute complex actin or microtubule-based phenomena. For example, a number of extracts support actin-dependent propulsion of Listeria monocytogenes, whereas Xenopus laevis extracts support formation of a microtubule-based meiotic spindle. Working in vitro opens these complex processes to biochemical dissection. Here, we describe methods to view these in vitro preparations by thin-section electron microscopy.

CRMP-1 enhances EVL-mediated actin elongation to build lamellipodia and the actin cortex.

Cells can control actin polymerization by nucleating new filaments or elongating existing ones. We recently identified CRMP-1 as a factor that stimulates the formation of Listeria monocytogenes actin comet tails, thereby implicating it in actin assembly. We now show that CRMP-1 is a major contributor to actin assembly in epithelial cells, where it works with the Ena/VASP family member EVL to assemble the actin cytoskeleton in the apical cortex and in protruding lamellipodia. CRMP-1 and EVL bind to one another and together accelerate actin filament barbed-end elongation. CRMP-1 also stimulates actin assembly in the presence of VASP and Mena in vitro, but CRMP-1-dependent actin assembly in MDCK cells is EVL specific. Our results identify CRMP-1 as a novel regulator of actin filament elongation and reveal a surprisingly important role for CRMP-1, EVL, and actin polymerization in maintaining the structural integrity of epithelial sheets.