The Actin Cytoskeleton in Myelinating Cells

Neurochemical Research - Myelinating cells of both the peripheral and central nervous systems (CNSs) undergo dramatic cytoskeletal reorganization in order to differentiate and produce myelin....     

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Extracellular Components Implicated in the Stationary Organization of the Actin Cytoskeleton in Mesophyll Cells of Vallisneria

In mesophyll cells of Vallisneria gigantea Graebner, an aquaticangiosperm, the association of the plasma membrane with thecell wall at the end wall has been reported to be indispensablefor the mechanism that maintains the stationary organizationof the bundles of microfilaments (MFs) [Masuda et al. (1991)Protoplasma 162: 151]. To identify putative extracellular componentsthat might play a crucial role in this mechanism, we examinedthe effects of two exogenously applied synthetic hexapeptides,GRGDSP and ARYDEI, which include an RGD and an RYD motif, respectively.The RGD motif is known as a recognition site in molecules requiredfor adhesion to the substratum at sites of focal contacts. Within24 h, both peptides (at concentrations of 1–15 mM) inducedextremely abnormal patterns of cytoplasmic streaming, as wellas the striking disruption of the arrangement of bundles ofMFs. GRGESP and ARYEEI peptides, used as controls, had no detectableeffects. Immunofluorescence microscopy revealed that polyclonalantibodies against the ARYDEI peptide bound to the cell wallsof mesophyll cells while a preimmune serum did not. Westernblotting analysis demonstrated that the antibodies recognizedpolypeptides of 54 kDa and 27 kDa in an extract of total proteinsfrom the leaves of Vallisneria. The results suggest that someextracellular protein(s), with a conserved RGD or RYD motifin its amino acid sequence, might be involved in the maintenanceof the stationary organization of the bundles of MFs. (Received August 13, 1996; Accepted January 23, 1997)     

Role of Actin Cytoskeleton in Regulation of Ion Transport: Examples from Epithelial Cells

The identification of molecular water transporters and the generation of transgenic mice lacking water transporting proteins has created a need for accurate methods to measure water permeability. This review is focused on methodology to characterize water permeability in living cells and complex multicellular tissues. The utility of various parameters defining water transport is critically evaluated, including osmotic water permeability (P _f ), diffusional water permeability (P _d ), Arrhenius activation energies (E _a ), and solute reflection coefficients (σ _p ). Measurements in cellular and complex tissues can be particularly challenging because of uncertainties in barrier geometry and surface area, heterogeneity in membrane transporting properties, and unstirred layer effects. Strategies to measure plasma membrane P _f in cell layers are described involving light scattering, total internal reflection fluorescence microscopy, confocal microscopy, interferometry, spatial filtering microscopy, and volume-sensitive fluorescent indicators. Dye dilution and fluorescent indicator methods are reviewed for measurement of P _f across cell and tissue barriers. Novel fluorescence and gravimetric methods are described to quantify microvascular and epithelial water permeabilities in intact organs, using as an example lungs from aquaporin knockout mice. Finally, new measurement strategies and applications are proposed, including high-throughput screening for identification of aquaporin inhibitors. Received: 3 August 1999/Revised: 22 September 1999     

Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K⁺, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca²⁺ influx through L-type Ca²⁺ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca²⁺ influx and accelerated F-actin breakdown on removal of Ca²⁺.To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca²⁺ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

Actin Out: Regulation of the Synaptic Cytoskeleton

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.     

Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

Rotavirus infection induces an increase in [Ca²⁺]_cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca²⁺ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca²⁺ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.     

A Morphogenesis Checkpoint Monitors the Actin Cytoskeleton in Yeast

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739– 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657– 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.     

The Polymeric State of Actin in the Human Erythrocyte Cytoskeleton

Reports on the polymeric state of actin in the red cell have been diverse. We have used phalloidin to stabilize the actin in erythrocyte ghosts prior to extraction in low ionic strength media. A mild proteolytic digestion and Sepharose 4B gel filtration enable an F-actin polymer to be isolated in pure form [1]. Detailed size analysis of this polymer in a range of experiments suggests that actin exists in the erythrocyte principally as a polymer of 100 nm length composed of 30 monomers in a double helical chain 15 monomers long with an estimated molecular weight of 1.3 × 10⁶ daltons.     

Homocysteine Induces Hypophosphorylation of Intermediate Filaments and Reorganization of Actin Cytoskeleton in C6 Glioma Cells

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 μM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 μM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 μM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 μM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 μM Hcy but only slightly attenuated the effect induced by of 500 μM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.     

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.     

Co-sedimentation of Actin, Tubulin andMembranes in the Cytoskeleton Fractions from Peas and Mouse 3T3 Cells

Pea stem tissue (Pisum sativum L. var. Alaska) was homogenizedin a recently-developed cytoskeleton-stabilizing buffer, CSB,(Abeand Da vies, 1991) and homogenates electrophoresed and blottedon to membranes. Blots probed individually withantibodies toactin, alpha-tubulin, and beta-tubulin, revealed bands withapparent molecular weights of 42, 46, and 48–50 kDa,respectively.Blots probed with all three antibodies simultaneously revealedall three bands which could be distinguished in thesame lane.Homogenates of mouse 3T3 cells yielded an actin band at about42 kDa, but both alpha- and beta-tubulin appeared atabout 50kDa and thus could not be distinguished on blots probed simultaneously.This ‘triple-blotting technique’ was, therefore,suitablefor pea tissue, but not for mouse tissue. In pea tissue, sedimentabletubulin and actin were found maximally in the 4000 xg pelletand less in successive 15000 and l00000xg pellets. Both EGTAand Mg²⁺ which had been found earlier to beessential for stabilityof the actin cytoskeleton as revealed by fluorescence microscopy,were essential for co-sedimentation of actinand tubulin. Incontrast to the results with pea stems, only the actin componentof the cytoskeleton could be isolated from mouse 3T3 cells usingCSB. Pea tissue was homogenized in CSB without PTE and the resultingcytoskeletal pellets resuspended in actin- or tubulin-solubilizingbuffers with and without PTE. In the absence of PTE, the bufferssolubilized their appropriate cytoskeletal protein, but littleof the other protein, while in the presence of PTE both proteinswere quite effectively solubilized by both buffers. Incontrast,in CSB with or without PTE, both proteins remained in the sedimentablefraction. These results, taken together withother evidence,indicate that microtubules, as well as microfilaments are importantcomponents of the sedimentable cytoskeletonfraction of peasand that the membrane system is intimately involved in organizationof the cytoskeleton in peas. Key words: Actin, tubulin, membranes, detergent, Ca²⁺, Mg²⁺, cytoskeleton     

mTORC2调节细胞骨架的信号通路

近几年来,关于哺乳动物雷帕霉素靶（mammalian target of rapamycin,mTOR）在各种哺乳动物细胞中调节肌动蛋白微丝极化及肌球蛋白微丝网形成的研究一直在不断地取得新的进展。尽管到目前为止,包括mTORC2上游和下游在内的相关的调控路径还未明确,但是因为mTORC6,的物学多样性,使其成为了当今生物学研究的焦点之一。基于长久以来特别是近五年对mTORC2的研究,在涉及细胞运动迁移、增殖分化、蛋白质合成、凋亡及自噬等生物学功能的研究中,一些重要的下游相关调控分子和蛋白相继被发现,比如P—Rexl／2、Rho家族GTPases、PKC、cAMP、p27kip1等。该综述着重总结了mTORC2实现这些生物学功能所可能通过的四条路径。当然,仍然需要大量的实验数据和研究证据进一步地证实和完善这些已经发现的可能存在的路径。