Search

The transport of macromolecules into the cell nucleus occurs through nuclear pore complexes (NPCs) and is mediated by cellular receptors. Recently, a novel mechanism of nuclear entry, in which actin polymerization provides a propulsive force driving the transport through the NPC, has been proposed. This mechanism is used by the nucleocapsid from baculovirus, one of the largest viruses to replicate in the nucleus of their host cells, which crosses the NPC and enters the nucleus independently of cellular receptors. The baculovirus nucleocapsid contains a protein that hijacks the cellular actin polymerization machinery to assemble actin filaments that propel the nucleocapsid through the host cell cytoplasm. In this study, we functionalized carbon nanotubes by covalently attaching a protein domain responsible for inducing actin polymerization and investigated their nuclear entry. We found that the functionalized carbon nanotubes were able to enter the cell nucleus under permissive conditions for actin polymerization, but not when this process was inhibited. We conclude that the mechanical force generated by actin polymerization can drive cargo entry into the cell nucleus. Our results support a novel force-driven mechanism for molecular entry into the cell nucleus. [ABSTRACT FROM AUTHOR]

Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction. [ABSTRACT FROM AUTHOR]

Plasmodium actins form very short filaments and have a noncanonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths have remained unknown. Using high-resolution crystallography, we show that magnesium binding causes a slight flattening of the Plasmodium actin I monomer, and subsequent phosphate release results in a more twisted conformation. Thus, the Mg-bound monomer is closer in conformation to filamentous (F) actin than the Ca form, and this likely facilitates polymerization. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A loop. Asp180 interacts with either Lys270 or His74, depending on the protonation state of the histidine, while Arg178 links the inner and outer domains (ID and OD) of the actin protomer. Hence, the A loop acts as a switch between stable and unstable filament conformations, the latter leading to fragmentation. Our data provide a comprehensive model for polymerization, ATP hydrolysis and phosphate release, and fragmentation of parasite microfilaments. Similar mechanisms may well exist in canonical actins, although fragmentation is much less favorable due to several subtle sequence differences as well as the methylation of His73, which is absent on the corresponding His74 in Plasmodium actin I. [ABSTRACT FROM AUTHOR]

Serum response factor and its cofactor myocardin-related transcription factor (MRTF) are key elements of muscle-mass adaptation to workload. The transcription of target genes is activated when MRTF is present in the nucleus. The localization of MRTF is controlled by its binding to G-actin. Thus, the pathway can be mechanically activated through the mechanosensitivity of the actin cytoskeleton. The pathway has been widely investigated from a biochemical point of view, but its mechanical activation and the timescales involved are poorly understood. Here, we applied local and global mechanical cues to myoblasts through two custom-built set-ups, magnetic tweezers and stretchable substrates. Both induced nuclear accumulation of MRTF-A. However, the dynamics of the response varied with the nature and level of mechanical stimulation and correlated with the polymerization of different actin sub-structures. Local repeated force induced local actin polymerization and nuclear accumulation of MRTF-A by 30 minutes, whereas a global static strain induced both rapid (minutes) transient nuclear accumulation, associated with the polymerization of an actin cap above the nucleus, and long-term accumulation, with a global increase in polymerized actin. Conversely, high strain induced actin depolymerization at intermediate times, associated with cytoplasmic MRTF accumulation. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 μM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins 21-activated kinase (PAK) nteracting xchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation. [ABSTRACT FROM AUTHOR]

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson disease. Genetics and neuropathology link Parkinson disease with the microtubule-binding protein tau, but the mechanism of action of LRRK2 mutations and the molecular connection between tau and Parkinson disease are unclear. Here, we investigate the interaction of LRRK and tau in Drosophila and mouse models of tauopathy. We find that either increasing or decreasing the level of fly Lrrk enhances tau neurotoxicity, which is further exacerbated by expressing Lrrk with dominantly acting Parkinson disease—associated mutations. At the cellular level, altering Lrrk expression promotes tau neurotoxicity via excess stabilization of filamentous actin (F-actin) and subsequent mislocalization of the critical mitochondrial fission protein dynamin-1-like protein (Drp1). Biochemically, monomeric LRRK2 exhibits actin-severing activity, which is reduced as increasing concentrations of wild-type LRRK2, or expression of mutant forms of LRRK2 promote oligomerization of the protein. Overall, our findings provide a potential mechanistic basis for a dominant negative mechanism in LRRK2-mediated Parkinson disease, suggest a common molecular pathway with other familial forms of Parkinson disease linked to abnormalities of mitochondrial dynamics and quality control, and raise the possibility of new therapeutic approaches to Parkinson disease and related disorders. [ABSTRACT FROM AUTHOR]

Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into “pedestals” beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP/Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir. [ABSTRACT FROM AUTHOR]

A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips. [ABSTRACT FROM AUTHOR]

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated. [ABSTRACT FROM AUTHOR]

Use of stem cell-based therapies in tissue engineering and regenerative medicine is hindered by efficient means of directed differentiation. For pluripotent stem cells, an initial critical differentiation event is specification to one of three germ lineages: endoderm, mesoderm, and ectoderm. Differentiation is known to be regulated by numerous extracellular and intracellular factors, but the role of the cytoskeleton during specification, or early differentiation, is still unknown. In these studies, we used agonists and antagonists to modulate actin polymerization and the actin-myosin molecular motor during spontaneous differentiation of embryonic stem cells in embryoid bodies. We found that inhibiting either actin polymerization or actin-myosin interactions led to a decrease in differentiation to the mesodermal lineage and an increase in differentiation to the endodermal lineage. Thus, targeting processes that regulate cytoskeletal tension may be effective in enhancing or inhibiting differentiation towards cells of the endodermal or mesodermal lineages, which include hepatocytes, islets, cardiomyocytes, endothelial cells, and osteocytes. Therefore, these fundamental findings demonstrate that modulation of the cytoskeleton may be useful in production for a range of cell-based therapies, including for liver, pancreatic, cardiac, vascular, and orthopedic applications. [ABSTRACT FROM AUTHOR]

Root-knot nematodes secrete effectors that manipulate their host plant cells so that the nematode can successfully establish feeding sites and complete its lifecycle. The root-knot nematode feeding structures, their “giant cells,” undergo extensive cytoskeletal remodeling. Previous cytological studies have shown the cytoplasmic actin within the feeding sites looks diffuse. In an effort to study root-knot nematode effectors that are involved in giant cell organogenesis, we have identified a nematode effector called MiPFN3 (Meloidogyne incognita Profilin 3). MiPFN3 is transcriptionally up-regulated in the juvenile stage of the nematode. In situ hybridization experiments showed that MiPFN3 transcribed in the nematode subventral glands, where it can be secreted by the nematode stylet into the plant. Moreover, Arabidopsis plants that heterologously expressed MiPFN3 were more susceptible to root-knot nematodes, indicating that MiPFN3 promotes nematode parasitism. Since profilin proteins can bind and sequester actin monomers, we investigated the function of MiPFN3 in relation to actin. Our results show that MiPFN3 suppressed the aberrant plant growth phenotype caused by the misexpression of reproductive actin (AtACT1) in transgenic plants. In addition, it disrupted actin polymerization in an in vitro assay, and it reduced the filamentous actin network when expressed in Arabidopsis protoplasts. Over a decade ago, cytological studies showed that the cytoplasmic actin within nematode giant cells looked fragmented. Here we provide the first evidence that the nematode is secreting an effector that has significant, direct effects on the plant’s actin cytoskeleton. [ABSTRACT FROM AUTHOR]

We present a Master Equation approach to calculating polymerization dynamics and force generation by branched actin networks at membranes. The method treats the time evolution of the F-actin distribution in three dimensions, with branching included as a directional spreading term. It is validated by comparison with stochastic simulations of force generation by actin polymerization at obstacles coated with actin “nucleation promoting factors” (NPFs). The method is then used to treat the dynamics of actin polymerization and force generation during endocytosis in yeast, using a model in which NPFs form a ring around the endocytic site, centered by a spot of molecules attaching the actin network strongly to the membrane. We find that a spontaneous actin filament nucleation mechanism is required for adequate forces to drive the process, that partial inhibition of branching and polymerization lead to different characteristic responses, and that a limited range of polymerization-rate values provide effective invagination and obtain correct predictions for the effects of mutations in the active regions of the NPFs. [ABSTRACT FROM AUTHOR]

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both halloidin post-fixation staining and ifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin odosome-ike clusters”. [ABSTRACT FROM AUTHOR]

To understand shapes and movements of cells undergoing lamellipodial motility, we systematically explore minimal free-boundary models of actin-myosin contractility consisting of the force-balance and myosin transport equations. The models account for isotropic contraction proportional to myosin density, viscous stresses in the actin network, and constant-strength viscous-like adhesion. The contraction generates a spatially graded centripetal actin flow, which in turn reinforces the contraction via myosin redistribution and causes retraction of the lamellipodial boundary. Actin protrusion at the boundary counters the retraction, and the balance of the protrusion and retraction shapes the lamellipodium. The model analysis shows that initiation of motility critically depends on three dimensionless parameter combinations, which represent myosin-dependent contractility, a characteristic viscosity-adhesion length, and a rate of actin protrusion. When the contractility is sufficiently strong, cells break symmetry and move steadily along either straight or circular trajectories, and the motile behavior is sensitive to conditions at the cell boundary. Scanning of a model parameter space shows that the contractile mechanism of motility supports robust cell turning in conditions where short viscosity-adhesion lengths and fast protrusion cause an accumulation of myosin in a small region at the cell rear, destabilizing the axial symmetry of a moving cell. [ABSTRACT FROM AUTHOR]

Leiomodin proteins are vertebrate homologues of tropomodulin, having a role in the assembly and maintenance of muscle thin filaments. Leiomodin2 contains an N-terminal tropomodulin homolog fragment including tropomyosin-, and actin-binding sites, and a C-terminal Wiskott-Aldrich syndrome homology 2 actin-binding domain. The cardiac leiomodin2 isoform associates to the pointed end of actin filaments, where it supports the lengthening of thin filaments and competes with tropomodulin. It was recently found that cardiac leiomodin2 can localise also along the length of sarcomeric actin filaments. While the activities of leiomodin2 related to pointed end binding are relatively well described, the potential side binding activity and its functional consequences are less well understood. To better understand the biological functions of leiomodin2, in the present work we analysed the structural features and the activities of Rattus norvegicus cardiac leiomodin2 in actin dynamics by spectroscopic and high-speed sedimentation approaches. By monitoring the fluorescence parameters of leiomodin2 tryptophan residues we found that it possesses flexible, intrinsically disordered regions. Leiomodin2 accelerates the polymerisation of actin in an ionic strength dependent manner, which relies on its N-terminal regions. Importantly, we demonstrate that leiomodin2 binds to the sides of actin filaments and induces structural alterations in actin filaments. Upon its interaction with the filaments leiomodin2 decreases the actin-activated Mg2+-ATPase activity of skeletal muscle myosin. These observations suggest that through its binding to side of actin filaments and its effect on myosin activity leiomodin2 has more functions in muscle cells than it was indicated in previous studies. [ABSTRACT FROM AUTHOR]

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. [ABSTRACT FROM AUTHOR]

Chondrocytes are known to be physiologically loaded with diverse physical factors such as compressive stress, shear stress and hydrostatic pressure. Although the effects of those mechanical stimuli onto various cell models have been widely studied, those of hypergravity have not yet been revealed clearly. Hereby, we hypothesized that the hypergravity affects relative positions of intracellular elements including nucleus and cytoskeletons due to their density differences, triggering mechanotransduction in the cell. The aim of this study was to investigate the effect of hypergravity on c-fos expression in the murine ATDC5 chondroprogenitor cells, as c-fos is a well known key regulator of cell proliferation and differentiation, including in chondrocytes. We first found that hypergravity down-regulated c-fos expression transiently via ROCK/Rho-GTP and PI3K signaling, and the down-regulation was suppressed by inhibition of actin polymerization. [ABSTRACT FROM AUTHOR]

Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes. [ABSTRACT FROM AUTHOR]

Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300–400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression. [ABSTRACT FROM AUTHOR]

As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell–specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization. [ABSTRACT FROM AUTHOR]

Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. [ABSTRACT FROM AUTHOR]

Primary cilia are non-motile cilia that serve as cellular antennae for sensing and transducing extracellular signals. In general, primary cilia are generated by cell quiescence signals. Recent studies have shown that manipulations to increase actin assembly suppress quiescence-induced ciliogenesis. To further examine the role of actin dynamics in ciliogenesis, we analyzed the effect of jasplakinolide (Jasp), a potent inducer of actin polymerization, on ciliogenesis. Unexpectedly, Jasp treatment induced ciliogenesis in serum-fed cells cultured at low density. In contrast, Jasp had no apparent effect on ciliogenesis in cells cultured at higher densities. Jasp-induced ciliogenesis was correlated with a change in cell morphology from a flat and adherent shape to a round and weakly adherent one. Jasp treatment also induced the phosphorylation and cytoplasmic localization of the YAP transcriptional co-activator and suppressed cell proliferation in low density-cultured cells. Overexpression of an active form of YAP suppressed Jasp-induced ciliogenesis. These results suggest that Jasp induces ciliogenesis through cell rounding and cytoplasmic localization and inactivation of YAP. Knockdown of LATS1/2 only faintly suppressed Jasp-induced YAP phosphorylation, indicating that LATS1/2 are not primarily responsible for Jasp-induced YAP phosphorylation. Furthermore, overexpression of active Src kinase suppressed Jasp-induced cytoplasmic localization of YAP and ciliogenesis, suggesting that down-regulation of Src activity is involved in Jasp-induced YAP inactivation and ciliogenesis. Our data suggest that actin polymerization does not suppress ciliogenesis per se but rather that cell rounding and reduced cell adhesion are more crucially involved in Jasp-induced ciliogenesis. [ABSTRACT FROM AUTHOR]

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. [ABSTRACT FROM AUTHOR]

Vascular regression is essential to remove redundant vessels during the formation of an efficient vascular network that can transport oxygen and nutrient to every corner of the body. However, no mechanism is known to explain how major blood vessels regress during development. Here we use the dorsal part of the caudal vein plexus (dCVP) in Zebrafish to investigate the mechanism of regression and discover a new role of Yap/Taz in vascular regression. During regression, Yap/Taz is activated by blood circulation in the endothelial cells. This leads to induction of Ctgf and actin polymerization. Interference with Yap/Taz activation decreased Ctgf production, which decreased actin polymerization and vascular regression. These results implicate a novel role of Yap/Taz in vascular regression. [ABSTRACT FROM AUTHOR]

Background and objectives: Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration. Methods: VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity. Results: Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor. Conclusions: Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration. [ABSTRACT FROM AUTHOR]

Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by non-muscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers–ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes. [ABSTRACT FROM AUTHOR]

Kidney podocytes’ function depends on finger-like projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling, lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen. [ABSTRACT FROM AUTHOR]

Background: During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. Methods: We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. Results: TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D LSCM and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. Conclusion: B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection. [ABSTRACT FROM AUTHOR]

Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus. [ABSTRACT FROM AUTHOR]

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. [ABSTRACT FROM AUTHOR]

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. [ABSTRACT FROM AUTHOR]

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. Arp2/3 is a well-characterised protein complex that can promote nucleation of new filaments, though its activity requires additional nucleation promotion factors (NPFs). The best recognized of these factors are the WASP family of proteins that contain binding motifs for both monomeric actin and for Arp2/3. Previously we demonstrated that the yeast WASP homologue, Las17, in addition to activating Arp2/3 can also nucleate actin filaments de novo, independently of Arp2/3. This activity is dependent on its polyproline rich region. Through biochemical and in vivo analysis we have now identified key motifs within the polyproline region that are required for nucleation and elongation of actin filaments, and have addressed the role of the WH2 domain in the context of actin nucleation without Arp2/3. We have also demonstrated that full length Las17 is able to bind liposomes giving rise to the possibility of direct linkage of nascent actin filaments to specific membrane sites to which Las17 has been recruited. Overall, we propose that Las17 functions as the key initiator of de novo actin filament formation at endocytic sites by nucleating, elongating and tethering nascent filaments which then serve as a platform for Arp2/3 recruitment and function. [ABSTRACT FROM AUTHOR]

Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. [ABSTRACT FROM AUTHOR]

For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility—the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. [ABSTRACT FROM AUTHOR]

Objective: Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells. Methodology: We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1. Results and Significance: We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes. [ABSTRACT FROM AUTHOR]

To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. [ABSTRACT FROM AUTHOR]

Objective: Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results: HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions: These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. [ABSTRACT FROM AUTHOR]

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. [ABSTRACT FROM AUTHOR]

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways. [ABSTRACT FROM AUTHOR]

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200–312 and 400–658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5). S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL) did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM) did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000–10000 nM) through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts. [ABSTRACT FROM AUTHOR]

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. [ABSTRACT FROM AUTHOR]