Paraquat induces irreversible actin cytoskeleton disruption in cultured human lung cells

The herbicide paraquat (PQ) induces the selective necrosis of type I and type II alveolar pneumocytes. We investigated the effect of PQ on human lung A549 cells to determine the possible role of cytoskeleton in lung cytotoxicity. At 80 mol/L PQ, a concentration that did not affect cell viability, the organization of actin cytoskeleton network depended on incubation time with the herbicide. Microfilaments appeared less numerous in 30% of the cells treated for 1 h. After 24 h, all the treated cells displayed only short filaments in the periphery. The effect of PQ on actin cytoskeleton was irreversible. Moreover, no modification of microtubule network was observed in PQ-treated cells. Next, we studied the effect of PQ on Chang Liver, an epithelial cell line from human liver. These cells appeared less sensitive to the herbicide than A549, and no cytoskeletal alteration was observed. To verify whether actin filament modifications in A549 cells were related to intracellular alterations of ATP concentrations, nucleotide levels during incubation with PQ were determined. The intracellular levels of ATP were not different in control and treated cells. Our results indicate that PQ induces specifically an irreversible actin filament disorganization on A549 cells and that the observed effect is independent of intracellular concentration of ATP.Abbreviations BSA bovine serum albumin - IC₅₀ concentration that produces 50% inhibitiition - PBS phosphate-buffered saline - PQ paraquat, 1,1-dimethyl-4,4-bipyridinium dichloride - SE standard error of the mean     

Phosphorylation-dependent cleavage of p130cas in apoptotic rat-1 cells

We previously demonstrated caspase-mediated cleavage of p130cas during apoptosis and identified two caspase-3 cleavage sites [1]. In this study, we investigated the phosphorylation-dependent cleavage of p130cas in apoptotic Rat-1 fibroblast cells. Lysophosphatidic acid and fibronectin induced p130cas phosphorylation, which in turn resulted in resistance to caspase-mediated cleavage. Alternatively, dephosphorylation by calf intestinal alkaline phosphatase, PP1, and LAR stimulated cleavage of p130cas by caspase-3, generating a 31-kDa fragment. During apoptosis, p130cas dephosphorylation seems to precede its cleavage. The phosphorylation of tyrosine and serine residues immediately adjacent to the two cleavage sites (DVPD(416) and DSPD(748)) strongly affected p130cas cleavage by caspase-3, both in vitro and in vivo. Furthermore, the generation of the 31-kDa cleavage fragment was strongly regulated by phosphorylation of a tyrosine residue at position 751 (DSPD(748) and GQY(751)). Our results collectively suggest that degradation of p130cas during apoptosis is modulated in a phosphorylation-dependent manner.     

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.     

Effect of actin cytoskeleton disruption on electric pulse‐induced apoptosis and electroporation in tumour cells

Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse‐induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.     

EhRabB mobilises the EhCPADH complex through the actin cytoskeleton during phagocytosis of Entamoeba histolytica

Movement and phagocytosis are clue events in colonisation and invasion of tissues by Entamoeba histolytica, the protozoan causative of human amoebiasis. During phagocytosis, EhRab proteins interact with other functional molecules, conducting them to the precise cellular site. The gene encoding EhrabB is located in the complementary chain of the DNA fragment containing Ehcp112 and Ehadh genes, which encode for the proteins of the EhCPADH complex, involved in phagocytosis. This particular genetic organisation suggests that the three corresponding proteins may be functionally related. Here, we studied the relationship of EhRabB with EhCPADH and actin during phagocytosis. First, we obtained the EhRabB 3D structure to carry out docking analysis to predict the interaction sites involved in the EhRabB protein and the EhCPADH complex contact. By confocal microscopy, transmission electron microscopy, and immunoprecipitation assays, we revealed the interaction among these proteins when they move through different vesicles formed during phagocytosis. The role of the actin cytoskeleton in this event was also confirmed using Latrunculin A to interfere with actin polymerisation. This affected the movement of EhRabB and EhCPADH, as well as the rate of phagocytosis. Mutant trophozoites, silenced in EhrabB gene, evidenced the interaction of this molecule with EhCPADH and strengthened the role of actin during erythrophagocytosis.     

Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion

This study evaluated whether cytoskeletal alterations during the ischemic conditions associated with kidney preservation could determine apoptosis. Cytoskeletal alterations are among the main effects of ischemia and may induce apoptosis. Rat kidneys were preserved in University of Wisconsin (UW) solution for 24 h. Some groups of animals underwent 45 min of warm ischemia (WI) to evaluate its effect on both the actin cytoskeleton and apoptosis (assessed by caspase-3 activity and TUNEL staining). Swinholide A (SwinA) and Latrunculin B (LB), two actin cytoskeleton-targeted agents, were administered to assess the effect of direct actin disruption on apoptosis. Jasplakinolide (JP), a compound that stabilizes actin filaments, was administered to evaluate the effect of actin stabilization. Apoptosis was evaluated at 3 h of ex vivo reperfusion using the isolated perfused rat kidney (IPK) model. Results: Apoptosis increased during reperfusion with WI or administration of actin disruptor agents. Administration of stabilizing agents reversed apoptosis in kidneys that had previously undergone WI or had received an actin disruptor agent. Conclusion: The disruption of the actin cytoskeleton during ischemic conditions associated with kidney preservation induces apoptosis upon reperfusion through caspase-3 activation.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.     

Single cell rigidity sensing: A complex relationship between focal adhesion dynamics and large-scale actin cytoskeleton remodeling

ABSTRACT

Many physiological and pathological processes involve tissue cells sensing the rigidity of their environment. In general, tissue cells have been shown to react to the stiffness of their environment by regulating their level of contractility, and in turn applying traction forces on their environment to probe it. This mechanosensitive process can direct early cell adhesion, cell migration and even cell differentiation. These processes require the integration of signals over time and multiple length scales. Multiple strategies have been developed to understand force- and rigidity-sensing mechanisms and much effort has been concentrated on the study of cell adhesion complexes, such as focal adhesions, and cell cytoskeletons. Here, we review the major biophysical methods used for measuring cell-traction forces as well as the mechanosensitive processes that drive cellular responses to matrix rigidity on 2-dimensional substrates.     

Discordance between the effect of modulators of calcium on staurosporine-induced apoptosis and staurosporine-induced actin degradation

Staurosporine produced DNA fragmentation characteristic of apoptosis and a dramatic alteration of cell structure that include alterations of cytoskeletal actin and cytoplasmic condensation with vacuolization. These changes were not induced by chelerythrine, a more specific PKC inhibitor than staurosporine. The calcium chelator, BAPTA, significantly reduced staurosporine-induced DNA fragmentation but did not affect staurosporine-induced changes in cytoskeletal actin. These data suggest that DNA fragmentation and actin degradation in apoptosis, induced by staurosporine, are under different regulatory control by calcium.     

Interaction of the NG2 proteoglycan with the actin cytoskeleton

The NG2 chondroitin sulfate proteoglycan is a membrane-spanning molecule expressed by immature precursor cells in a variety of developing tissues. In tightly adherent cell lines with a flattened morphology, NG2 is organized on the cell surface in linear arrays that are highly co-localized with actin and myosin-containing stress fibers in the cytoskeleton. In contrast, microtubules and intermediate filaments in the cytoskeleton exhibit completely different patterns of organization, suggesting that NG2 may use microfilamentous stress fibers as a means of cytoskeletal anchorage. Consistent with this is the observation that cytochalasin D disrupts the organization of both stress fibers in the cytoskeleton and NG2 on the cell surface. Very similar linear cell surface arrays are also seen with three other cell surface molecules thought to interact with the actin cytoskeleton: the α₅β₁ integrin, the CD44 proteoglycan, and the L1 neuronal cell adhesion molecule. Since the cytoplasmic domains of these four molecules are dissimilar, it seems possible that cytoskeletal anchorage in each case may occur via different mechanisms. One indication of such differences can be seen in colchicine-treated cells which have lost their flattened morphology but still retain long actin-positive tendrils as remnants of the actin cytoskeleton. NG2 and α₅β₁ are associated with these tendrils while CD44 and L1 are not, suggesting that at least two subclasses of cell surface molecules exist which can interact with different subdomains of the actin cytoskeleton. © 1996 Wiley-Liss, Inc.     

Interaction of plant polysomes with the actin cytoskeleton

Protein composition and functional activity of various polysome subpopulations isolated from Vicia faba L. leaves and Triticum aestivum L. and Hordeum vulgare L. seedlings were studied. Membrane- and cytoskeleton-bound polysomes were more active in the wheat germ cell-free translational system than free polysomes. Several non-ribosomal proteins were detected in the polysome preparations by gel electrophoresis and Western blot analysis: (1) a canonical actin of mol wt 42 kDa; (2) a 40 kDa protein, demonstrating affinity for ribosomes, sharing some determinants with actin, and present predominantly in the subpopulations of bound polysomes; and (3) an acidic ribosome-associated p40 evenly distributed between free and bound polysomes. The possibility of involvement of these proteins in interactions between polysomes and the actin cytoskeleton is discussed.     

Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death

The caspase family of proteases cleaves large number of proteins resulting in major morphological and biochemical changes during apoptosis. Yet, only a few of these proteins have been reported to selectively cleaved by caspase-2. Numerous observations link caspase-2 to the disruption of the cytoskeleton, although it remains elusive whether any of the cytoskeleton proteins serve as bona fide substrates for caspase-2. Here, we undertook an unbiased proteomic approach to address this question. By differential proteome analysis using two-dimensional gel electrophoresis, we identified four cytoskeleton proteins that were degraded upon treatment with active recombinant caspase-2 in vitro. These proteins were degraded in a caspase-2-dependent manner during apoptosis induced by DNA damage, cytoskeleton disruption or endoplasmic reticulum stress. Hence, degradation of these cytoskeleton proteins was blunted by siRNA targeting of caspase-2 and when caspase-2 activity was pharmacologically inhibited. However, none of these proteins was cleaved directly by caspase-2. Instead, we provide evidence that in cells exposed to apoptotic stimuli, caspase-2 probed these proteins for proteasomal degradation. Taken together, our results depict a new role for caspase-2 in the regulation of the level of cytoskeleton proteins during apoptosis.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C‐terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline‐rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell‐wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin‐dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin‐remodelling mechanisms.     

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.