F-actin Network Research Articles

A Mechanochemical Model of Actin Filaments

In eukaryotic cells, actin filaments are involved in important processes such as motility, division, cell shape regulation, contractility, and mechanosensation. Actin filaments are polymerized chains of monomers, which themselves undergo a range of chemical events such as ATP hydrolysis, polymerization, and depolymerization. When forces are applied to F-actin, in addition to filament mechanical deformations, the applied force must also influence chemical events in the filament. We develop an intermediate-scale model of actin filaments that combines actin chemistry with filament-level deformations. The model is able to compute mechanical responses of F-actin during bending and stretching. The model also describes the interplay between ATP hydrolysis and filament deformations, including possible force-induced chemical state changes of actin monomers in the filament. The model can also be used to model the action of several actin-associated proteins, and for large-scale simulation of F-actin networks. All together, our model shows that mechanics and chemistry must be considered together to understand cytoskeletal dynamics in living cells.

Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E∗) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E∗. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E∗ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was demonstrated in this study, with positive links to cytoskeletal changes. The estradiol associated changes in osteoblast mechanical properties could bear implications for bone remodeling and its mechanical integrity.

Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F‐actin regulation

Annexin A2 (ANXA2) is present in vivo in the mid- and late-secretory endometria and is mainly localized in the luminal epithelium. Our aim was to evaluate its function in regulating the human implantation process. With an in vitro adhesion model, constructed to evaluate how the mouse embryo and JEG-3 spheroids attach to human endometrial epithelial cells, we demonstrated that ANXA2 inhibition significantly diminishes embryo adhesiveness. ANXA2 is also implicated in endometrial epithelial cell migration and trophoblast outgrowth. ANXA2 was seen to be linked to the RhoA/ROCK pathway and to regulate cell adhesion. We noted that ANXA2 inhibition significantly reduces active RhoA, although RhoA inactivation does not alter the ANXA2 levels. RhoA inactivation and ROCK inhibition also moderate embryo adhesiveness to endometrial epithelial cells. We corroborated that the induction of constitutively active RhoA partially reverses the effects of ANXA2 inhibition on endometrial adhesiveness. These molecules colocalize on the plasma membrane of endometrial epithelial cells, and a large proportion of ANXA2 and RhoA are colocalized in the F-actin networks. The functional effects of ANXA2 inhibition and RhoA/ROCK inactivation are associated with significant alterations in F-actin organization and its depolymerization. ANXA2 may act upstream of the RhoA/ROCK pathway by regulating F-actin remodeling and is a key factor in human endometrial adhesiveness.

The Role of Munc18-1 and Its Orthologs in Modulation of Cortical F-Actin in Chromaffin Cells

Munc18-1 was originally described as an essential docking factor in chromaffin cells. Recent findings showed that Munc18-1 has an additional role in the regulation of the cortical F-actin network, which is thought to function as a physical barrier preventing secretory vesicles from access to their release sites under resting conditions. In our review, we discuss whether this function is evolutionarily conserved in all Sec1/Munc18-like (SM) proteins. In addition, we introduce a new quantification method that improves the analysis of cortical filamentous actin (F-actin) in comparison with existing methods. Since the docking process is highly evolutionarily conserved in the SM protein superfamily, we use our novel quantification method to investigate whether the F-actin-regulating function is similarly conserved among SM proteins. Our preliminary data suggest that the regulation of cortical F-actin is a shared function of SM proteins, and we propose a way to gain more insight in the molecular mechanism underlying the Munc18-1-mediated cortical F-actin regulation.

Integrin-linked kinase at a glance

Integrins are a large family of cell–extracellular matrix (ECM) and cell–cell adhesion molecules that regulate development and tissue homeostasis by controlling cell migration, survival, proliferation and differentiation ([Hynes, 2002][1]). They are non-covalently associated heterodimers

The Adaptor Protein Crk Controls Activation and Inhibition of Natural Killer Cells

Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16 and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-E alone induced Crk phosphorylation in NKG2A(+) NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.

F‐actin distribution at nodes of Ranvier and Schmidt‐Lanterman incisures in mammalian sciatic nerves

Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model.

The Mechanical Behavior of Mutant K14-R125P Keratin Bundles and Networks in NEB-1 Keratinocytes

Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.

An affine micro-sphere-based constitutive model, accounting for junctional sliding, can capture F-actin network mechanics

Actin filaments are a major component of the cytoskeleton and play a crucial role in cell mechanotransduction. F-actin networks can be reconstituted in vitro and their mechanical behaviour has been studied experimentally. Constitutive models that assume an idealised network structure, in combination with a non-affine network deformation, have been successful in capturing the elastic response of the network. In this study, an affine network deformation is assumed, in which we propose an alternative 3D finite strain constitutive model. The model makes use of a micro-sphere to calculate the strain energy density of the network, which is represented as a continuous distribution of filament orientations in space. By incorporating a simplified sliding mechanism at the filament-to-filament junctions, premature filament locking, inherent to affine network deformation, could be avoided. The model could successfully fit experimental shear data for a specific cross-linked F-actin network, demonstrating the potential of the novel model.

Phosphorylation of CRN2 by CK2 regulates F-actin and Arp2/3 interaction and inhibits cell migration.

CRN2 (synonyms: coronin 1C, coronin 3) functions in the re-organization of the actin network and is implicated in cellular processes like protrusion formation, secretion, migration and invasion. We demonstrate that CRN2 is a binding partner and substrate of protein kinase CK2, which phosphorylates CRN2 at S463 in its C-terminal coiled coil domain. Phosphomimetic S463D CRN2 loses the wild-type CRN2 ability to inhibit actin polymerization, to bundle F-actin, and to bind to the Arp2/3 complex. As a consequence, S463D mutant CRN2 changes the morphology of the F-actin network in the front of lamellipodia. Our data imply that CK2-dependent phosphorylation of CRN2 is involved in the modulation of the local morphology of complex actin structures and thereby inhibits cell migration.

Apico-basal elongation requires a drebrin-E–EB3 complex in columnar human epithelial cells

Although columnar epithelial cells are known to acquire an elongated shape, the mechanisms involved in this morphological feature have not yet been completely elucidated. Using columnar human intestinal Caco2 cells, it was established here that the levels of drebrin E, an actin-binding protein, increase in the terminal web both in vitro and in vivo during the formation of the apical domain. Drebrin E depletion was found to impair cell compaction and elongation processes in the monolayer without affecting cell polarity or the formation of tight junctions. Decreasing the drebrin E levels disrupted the normal subapical F-actin-myosin-IIB-βII-spectrin network and the apical accumulation of EB3, a microtubule-plus-end-binding protein. Decreasing the EB3 levels resulted in a similar elongation phenotype to that resulting from depletion of drebrin E, without affecting cell compaction processes or the pattern of distribution of F-actin-myosin-IIB. In addition, EB3, myosin IIB and βII spectrin were found to form a drebrin-E-dependent complex. Taken together, these data suggest that this complex connects the F-actin and microtubule networks apically during epithelial cell morphogenesis, while drebrin E also contributes to stabilizing the actin-based terminal web.

Lysozyme facilitates adherence of Enterococcus faecium to host cells and induction of necrotic cell death

The prevalence of infections with enterococci is increasing worldwide. However, little is known about the mechanisms which enable these opportunistic pathogens to cause infections of their host. Here we demonstrate that Enterococcus faecium in the presence of lysozyme induces necrosis in human and mouse cells after 4 h indicated by disrupted cellular membranes of epithelial (HeLa), myeloid (U937, J774A.1) and lymphoid (Jurkat J16, thymocytes), but not intestinal epithelial cells (CaCo-2, CMT-93). Using an appropriate mutant strain it was shown that the enterococcal surface-protein SgrA is involved in cell death induction in mouse cells (J774A.1, thymocytes). Microscopic analyses of epithelial cells 30 min post infection revealed that lysozyme increases adhesion of E. faecium to HeLa, but not CaCo-2 cells. At that time the phalloidin-FITC-stained cytoskeleton of infected cells was still intact, whereas 2 h post infection the F-actin network of HeLa, but not CaCo-2 cells was disrupted. Hence, the early, lysozyme-mediated increase of bacterial adherence plays an important role for cell death induction by E. faecium in HeLa cells. Moreover, bacterial extracellular hydrogen peroxide might contribute to necrosis induction, since the rate of propidium iodide-positive HeLa and J774A.1 cells was lowered after infection with a ROS-deficient E. faecium mutant.

NAB-1 instructs synapse assembly by linking adhesion molecules and F-actin to active zone proteins

During synaptogenesis, macromolecular protein complexes assemble at the pre- and postsynaptic membrane. Extensive literature identifies numerous transmembrane molecules sufficient to induce synapse formation and several intracellular scaffolding molecules responsible for assembling active zones and recruiting synaptic vesicles. However, little is known about the molecular mechanisms coupling membrane receptors to active zone molecules during development. Using C.elegans, we identify an F-actin network present at nascent presynaptic terminals required for presynaptic assembly. We unravel a sequence of events where specificity-determining adhesion molecules define the location of developing synapses and locally assemble F-actin. Next, an adaptor protein NAB-1/Neurabin binds to F-actin and recruits active zone proteins, SYD-1 and SYD-2/Liprin-α by forming a tripartite complex. NAB-1 localizes transiently to synapses during development and is required for presynaptic assembly. Together, we identify a role for the actin cytoskeleton during presynaptic development and characterize a molecular pathway where NAB-1 links synaptic partner recognition to active zone assembly.

Investigation of Molecular Level Stress-Strain Relationships in Systems of Entangled F-Actin by Combined Force-Measuring Optical Tweezers and Fluorescence Microscopy

Actin plays a major role in cell structure, cell motility, vesicle and organelle transport, and muscle contraction. Actin's ability to play these major roles is a direct consequence of the intricate relationship between stress and strain in a variety of filamentous actin (F-actin) networks. A thorough understanding of the unique stress-strain relationships in complex F-actin networks at the molecular-level is currently lacking despite the importance of such networks to fields such as biomimetic material engineering and cell biology. Here we develop novel single-molecule instrumentation that combines dual-trap force-measuring optical tweezers with fluorescence microscopy to enable simultaneous characterization of intermolecular forces and molecular dynamics within F-actin networks. This instrumentation is combined with a novel technique in which single F-actin “probes” are used to apply molecular-level strains and measure induced stress within entangled F-actin systems while the deformations and dynamics of surrounding fluorescent-labeled filaments are simultaneously imaged. Specifically, the dual-trap optical tweezers trap fluorescent-labeled, microsphere-conjugated probes and measure the force induced on the probe as it is moved through a network of selectively-labeled entangled F-actin by using a nanoprecison piezoelectric stage to move the sample chamber relative to the traps. Fluorescence microscopy is used simultaneously to image the dynamics of the moving probe as well as the surrounding labeled molecules subject to the applied strain. Specific bioconjugation of the fluorescent polystyrene microspheres to the ends of labeled F-actin is achieved by carbodiimide attachment of gelsolin to microspheres and combining gelsolin-coated microspheres with fluorescent phalloidin labeled actin. This powerful single-molecule technique allows simultaneous measurement of intermolecular forces and dynamics and deformations of single molecules, providing the much needed link between stress and strain at the molecular level in complex F-actin networks.

Assembly kinetics determine the architecture of α-actinin crosslinked F-actin networks

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks.

Stochastic rate-dependent elasticity and failure of soft fibrous networks

This work focuses on modeling the rate-sensitive stiffening-to-softening transition in fibrous architectures mimicking crosslinked fibrous actin (F-actin) networks induced by crosslink unbinding. Using finite element based discrete network (DN) modeling combined with stochastic crosslink scission kinetics, we correlate the microstructural damage evolution with the macroscopic stress–strain responses of these networks as a function of applied deformation rate. Simulations of multiple DN realizations for fixed filament density indicate that an incubation strain exists, which characterizes the minimum macroscopic deformation that a network should accrue before damage initiates. This incubation strain exhibits a direct relationship with the applied strain rate. Simulations predict that the critical damage fraction corresponding to colossal softening is quite low, which may be ascribed to the network non-affinity and filament reorientation. Furthermore, this critical fraction appears to be independent of applied strain rate. Based on these characteristics, we propose a phenomenological damage evolution law mimicking scission kinetics in an average sense. This law is embedded within an existing continuum model that is extended to include non-affine effects induced by filament bending.

Endocannabinoid-binding CB1 and TRPV1 receptors as modulators of sperm capacitation

Mammalian spermatozoa reach the ability to fertilize only after they complete a complex series of physical-chemical modification, the capacitation. Recently, the endocannabinoid-binding type-1cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) channel have been proposed to play a key role in the control of capacitation. In particular CB1, acting via a Gi protein/cAMP/PKA pathway, maintains low cAMP levels in early stages of post ejaculatory life of male gametes. By this way it promotes the maintenance of membrane stability, thus avoiding the premature fusion of plasma membrane (PM) and outer acrosome membrane (OAM), which is mandatory for the exocytosis of acrosome content. TRPV1, on the contrary, becomes active during the latest stages of capacitation, and allows the rapid increase in intracellular calcium concentration that leads to the removal of the F-actin network interposed between PM and OAM, leading to their fusion and, ultimately, to the acrosome reaction.

Non-Contact Microrheology of Monolayers and Membranes

Understanding the mechanics/rheology of surface monolayers and lipid bilayers is of fundamental biological importance. One technique used to explored these questions is membrane microrheology, in which the observed thermal fluctuations of a tracer particle in the monolayer is used to extract the rheological data. This technique is challenging for at least two reasons. On the one hand, in fragile monolayer systems the presence of the tracer can locally perturb the monolayer. On the other hand, in sufficiently stiff monolayers/membranes it has proved problematic to embed the particle in it. In this talk, we develop a noncontact microrheological approach to avoid these issues by exploring the effect membrane rheology on the thermal fluctuations of a bead in the fluid near the monolayer. Specifically, we develop the theory of the force response function of a spherical particle submerged below either a Langmuir monolayer or a lipid bilayer. We show that one can use the observed thermal fluctuations of that submerged particle to extract rheological properties of the essentially two-dimensional monolayer or membrane. We also present experimental results on application of this technique to surfactant monolayers and monolayers bound to an F-actin network on the aqueous side of the Langmuir monolayer.

Urocortins Improve Dystrophic Skeletal Muscle Structure and Function through Both PKA- and Epac-Dependent Pathways

In Duchenne muscular dystrophy, the absence of dystrophin causes progressive muscle wasting and premature death. Excessive calcium influx is thought to initiate the pathogenic cascade, resulting in muscle cell death. Urocortins (Ucns) have protected muscle in several experimental paradigms. Herein, we demonstrate that daily s.c. injections of either Ucn 1 or Ucn 2 to 3-week-old dystrophic mdx(5Cv) mice for 2 weeks increased skeletal muscle mass and normalized plasma creatine kinase activity. Histological examination showed that Ucns remarkably reduced necrosis in the diaphragm and slow- and fast-twitch muscles. Ucns improved muscle resistance to mechanical stress provoked by repetitive tetanizations. Ucn 2 treatment resulted in faster kinetics of contraction and relaxation and a rightward shift of the force-frequency curve, suggesting improved calcium homeostasis. Ucn 2 decreased calcium influx into freshly isolated dystrophic muscles. Pharmacological manipulation demonstrated that the mechanism involved the corticotropin-releasing factor type 2 receptor, cAMP elevation, and activation of both protein kinase A and the cAMP-binding protein Epac. Moreover, both STIM1, the calcium sensor that initiates the assembly of store-operated channels, and the calcium-independent phospholipase A(2) that activates these channels were reduced in dystrophic muscle by Ucn 2. Altogether, our results demonstrate the high potency of Ucns for improving dystrophic muscle structure and function, suggesting that these peptides may be considered for treatment of Duchenne muscular dystrophy.

Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins

In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer antibodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD.