Interfering with the connection between the nucleus and the cytoskeleton affects nuclear rotation, mechanotransduction and myogenesis

Mechanical stress controls a broad range of cellular functions. The cytoskeleton is physically connected to the extracellular matrix via integrin receptors, and to the nuclear lamina by the LINC complex that spans both nuclear membranes. We asked here how disruption of this direct link from the cytoskeleton to nuclear chromatin affects mechanotransduction. Fibroblasts grown on flexible silicone membranes reacted to cyclic stretch by nuclear rotation. This rotation was abolished by inhibition of actomyosin contraction as well as by overexpression of dominant-negative versions of nesprin or sun proteins that form the LINC complex. In an in vitro model of muscle differentiation, cyclic strain inhibits differentiation and induces proliferation of C2C12 myoblasts. Interference with the LINC complex in these cells abrogated their stretch-induced proliferation, while stretch increased p38 MAPK and NFkappaB phosphorylation and the transcript levels of myogenic transcription factors MyoD and myogenin. We found that the physical link from the cytoskeleton to the nuclear lamina is crucial for correct mechanotransduction, and that disruption of the LINC complex perturbs the mechanical control of cell differentiation.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

How HIV takes advantage of the cytoskeleton in entry and replication

The host cell cytoskeleton plays a key role in the life cycle of viral pathogens whose propagation depends on mandatory intracellular steps. Accordingly, also the human immunodeficiency virus type 1 (HIV-1) has evolved strategies to exploit and modulate in particular the actin cytoskeleton for its purposes. This review will recapitulate recent findings on how HIV-1 hijacks the cytoskeleton to facilitate entry into, transport within and egress from host cells as well as to commandeer communication of infected with uninfected bystander cells.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton

Summary Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Phosphoinositide 3-kinase C2β regulates RhoA and the actin cytoskeleton through an interaction with Dbl

The regulation of cell morphology is a dynamic process under the control of multiple protein complexes acting in a coordinated manner. Phosphoinositide 3-kinases (PI3K) and their lipid products are widely involved in cytoskeletal regulation by interacting with proteins regulating RhoGTPases. Class II PI3K isoforms have been implicated in the regulation of the actin cytoskeleton, although their exact role and mechanism of action remain to be established. In this report, we have identified Dbl, a Rho family guanine nucleotide exchange factor (RhoGEF) as an interaction partner of PI3KC2β. Dbl was co-immunoprecipitated with PI3KC2β in NIH3T3 cells and cancer cell lines. Over-expression of Class II phosphoinositide 3-kinase PI3KC2β in NIH3T3 fibroblasts led to increased stress fibres formation and cell spreading. Accordingly, we found high basal RhoA activity and increased serum response factor (SRF) activation downstream of RhoA upon serum stimulation. In contrast, the dominant-negative form of PI3KC2β strongly reduced cell spreading and stress fibres formation, as well as SRF response. Platelet-derived growth factor (PDGF) stimulation of wild-type PI3KC2β over-expressing NIH3T3 cells strongly increased Rac and c-Jun N-terminal kinase (JNK) activation, but failed to show similar effect in the cells with the dominant-negative enzyme. Interestingly, epidermal growth factor (EGF) and PDGF stimulation led to increased extracellular signal-regulated kinase (Erk) and Akt pathway activation in cells with elevated wild-type PI3KC2β expression. Furthermore, increased expression of PI3KC2β protected NIH3T3 from detachment-dependent death (anoikis) in a RhoA-dependent manner. Taken together, these findings suggest that PI3KC2β modulates the cell morphology and survival through a specific interaction with Dbl and the activation of RhoA.

Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.

Translocation of GPIb and Fc receptor gamma-chain to cytoskeleton in mucetin-activated platelets

Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Ezrin/radixin/moesin: Versatile controllers of signaling molecules and of the cortical cytoskeleton

Ezrin, radixin and moesin (ERM) proteins are widely distributed proteins located in the cellular cortex, in microvilli and adherens junctions. They feature an N-terminal membrane binding domain linked by an alpha-helical domain to the C-terminal actin-binding domain. In the dormant state, binding sites in the N-terminal domain are masked by interactions with the C-terminal region. The alpha-helical domain also contributes to masking of binding sites. A specific sequence of signaling events results in dissociation of these intramolecular interactions resulting in ERM activation. ERM molecules have been implicated in mediating actin-membrane linkage and in regulating signaling molecules. They are involved in cell membrane organization, cell migration, phagocytosis and apoptosis, and may also play cell-specific roles in tumor progression. Their precise involvement in these processes has yet to be elucidated.

Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation.

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

WASP plays a novel role in regulating platelet responses dependent on alphaIIbbeta3 integrin outside-in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent -yet- actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by alphaIIbbeta3 integrin outside-in signalling, we questioned whether its function might be linked to integrin. Agonist-induced alphaIIbbeta3 activation (PAC-1 binding) was normal for patient platelets, indicating normal integrin inside-out signalling. Inside-out signalling (fibrinogen, JON/A binding) was also normal for wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in responses. Another integrin outside-in dependent response, fibrin clot retraction, involving contraction of the post-aggregation actin cytoskeleton, was also decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signalling.