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The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase

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MOLECULAR AND CELLULAR BIOLOGY 2000年第15期20卷 5469-5478页

作者： Genot, EM Arrieumerlou, C Ku, G Burgering, BMT Weiss, A Kramer, IM Univ Bordeaux 1 Growth Factors & Differentiat Lab F-33405 Talence France Univ London Imperial Coll Sci Technol & Med Dept Immunol London W12 0NN England CERVI Lab Immunol Cellulaire CNRS UMR 7627 F-75013 Paris France Univ Calif San Francisco Howard Hughes Med Inst Dept Med Microbiol & Immunol San Francisco CA 94143 USA Univ Utrecht Physiol Chem Lab NL-3584 CG Utrecht Netherlands

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. Here we report that the small GTPase Rad is implicated in this pathway, connecting the receptor with the lipid kinase. We show that in Jurkat cells, activated forms of Rad or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rad but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutive active PI 3-kinase is unaffected when dominant negative Rad is coexpressed, placing Rad upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, we found that Rad acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. Our results reveal a previously unrecognized role of the GTPase Rad, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rad as a downstream transducer for Akt/PKB activation.

关键词：色酮类/药理学细胞骨架/代谢细胞骨架/超微结构酶激活酶抑制剂/药理学吗啉类/药理学突变磷酸肌醇3-激酶类/拮抗剂和抑制剂磷酸肌醇3-激酶类/遗传学磷酸肌醇3-激酶类/代谢磷酰化蛋白质丝氨酸苏氨酸激酶原癌基因蛋白质类/代谢原癌基因蛋白质c-akt 受体抗原 T细胞/免疫学受体抗原 T细胞/代谢 T淋巴细胞/代谢苏氨酸/代谢肿瘤细胞培养的 rac1GTP结合蛋白质/遗传学 rac1GTP结合蛋白质/代谢人类

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Abnormal Schwann cell-axon interactions in CMT neuropathies - The effects of mutant Schwann cells on the axonal cytoskeleton and regeneration-associated myelination

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CHARCOT-MARIE-TOOTH DISORDERS 1999年第1期883卷 415-426页

作者： Sahenk, Z Ohio State Univ Neuromuscular Ctr Dept Neurol Columbus OH 43210 USA

Preferential distal axonal loss is a common feature of Charcot-Marie-Tooth (CMT) hereditary neuropathies, The general hypothesis tested here is that the axonal loss in these disorders is due to perturbed Schwann cell-axon interactions resulting from primary Schwann genetic defects. The nerve xenograft model was used to study the local influence of mutant Schwann cells derived from patients on axonal properties and the regeneration-associated myelination process. Sural nerve segments from individuals with PMP22 duplications or deletions and point mutations, as well as Cx32 point mutations were grafted into cut ends of the sciatic nerve of nude mice and studied at different time intervals after grafting. CMT1A and CMTX xenografts examined at 16 weeks show that the nude mouse axone within the proximal part of grafts demonstrate a significant increase in axonal area with an increase in the neurofilament and membranous organelle density compared to distal graft and distal host segments. A preferential distal axonal loss associated with a perpetual axonal atrophy, degeneration, and axonal sprouting was observed over time with increasing frequency at 8 to 16 weeks. A distal summation of the pathology, as evidenced by a greater amount of fiber loss in the distal graft segments, was present similar to that observed in patients. These alterations were seen to a lesser extent in PMP22 deletion or point mutation xenografts and were not observed in controls. Collectively, these findings show that Schwann cells bearing the PMP22 or Cx32 genetic defects cause major perturbations in Schwann cell-axon interactions emphasizing the role of axonal component in the pathogenesis of hereditary neuropathies.

关键词：轴突/病理学轴突/生理学轴突/超微结构夏科-马里-图斯病/遗传学夏科-马里-图斯病/病理学夏科-马里-图斯病/病理生理学连接蛋白类/遗传学细胞骨架/病理学细胞骨架/生理学细胞骨架/超微结构基因复制小鼠裸髓磷脂蛋白质类/遗传学神经再生许旺细胞/病理学许旺细胞/生理学坐骨神经/生理学坐骨神经/超微结构腓肠神经/病理学腓肠神经/移植移植异种动物人类小鼠

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Membrane tether formation from blebbing cells

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BIOPHYSICAL JOURNAL 1999年第6期77卷 3363-3370页

作者： Dai, JW Sheetz, MP Duke Univ Med Ctr Dept Cell Biol Durham NC 27710 USA

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.

关键词：黏着性生物物理学细胞膜/生理学细胞膜/超微结构细胞极性细胞骨架/生理学细胞骨架/超微结构肾小管近端/生理学肾小管近端/超微结构激光黑色素瘤/病理生理学黑色素瘤/超微结构渗透压压力表面张力肿瘤细胞培养的动物人类兔

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Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions

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MOLECULAR AND CELLULAR BIOLOGY 2000年第8期20卷 2865-2873页

作者： Bouchard, MJ Dong, YZ McDermott, BM Lam, DH Brown, KR Shelanski, M Bellvé, AR Racaniello, VR Columbia Univ Coll Phys & Surg Dept Microbiol New York NY 10032 USA Columbia Univ Coll Phys & Surg Dept Pathol New York NY 10032 USA Columbia Univ Coll Phys & Surg Dept Anat & Cell Biol New York NY 10032 USA Columbia Univ Coll Phys & Surg Dept Urol New York NY 10032 USA Columbia Univ Coll Phys & Surg Ctr Reprod Sci New York NY 10032 USA

Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily, Nectin-2 is a component of cell-cell adherens junctions and interacts with 1-afadin, an F-actin-binding protein, Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile, The production by nectin-2(-/-) mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2(-/-) mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.

关键词：细胞黏附分子/生理学细胞核/病理学细胞核/生理学细胞核/超微结构细胞骨架/病理学细胞骨架/生理学细胞骨架/超微结构基因表达调控/生理学细胞连接/遗传学精子/病理学精子/生理学精子/超微结构动物人类男(雄)性小鼠大鼠

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Cytoskeletal integration in a highly ordered sensory epithelium in the organ of Corti: reponse to loss of cell partners in the Bronx waltzer mouse

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JOURNAL OF NEUROCYTOLOGY 1999年第12期28卷 1017-1034页

作者： Tucker, JB Mackie, JB Bussoli, TJ Steel, KP Univ St Andrews Sch Biol St Andrews KY16 9TS Fife Scotland MRC Inst Hearing Res Nottingham NG7 2RD England

This report is concerned with control of cell shaping, positioning, and cytoskeletal integration in a highly ordered cochlear neuroepithelium. It is largely based on investigations of events that occur during abnormal morphogenesis of the organ of Corti in the Bronx waltzer (bv/bv) mutant mouse. The organ's sensory hair cells and adjacent supporting cells ordinarily construct a spatially elaborate and supracellularly integrated cytoskeletal framework. Large microtubule bundles are connected to cytoskeletal components in neighbouring cells by actin-containing meshworks that link them to substantial arrays of adherens junctions. In bv/bv mice, degeneration and loss of most inner hair cells and outer pillar cells occurs during organ development. These cells flank each side of a row of inner pillar cells that respond by upregulating assembly of their actin-containing meshworks. This only occurs in surface regions where they no longer contact cell types involved in construction of the cytoskeletal framework. The meshworks are larger and exhibit a more extensive sub-surface deployment than is normally the case. Hence, assembly of intercellular cytoskeletal connecting components can proceed without contact with appropriate cell neighbours but termination of assembly is apparently subject to a negative feedback control triggered by successful completion of intercellular connection with the correct cell neighbours. In addition, inner pillar cells compensate for loss of cell neighbours by interdigitating and overlapping each other more extensively than is usually the case to increase opportunities for generating adherens junctions. Certain adherens junctions in the organs of +/+ and bv/bv mice exhibit features that distinguish them from all previously described cell junctions. The dense plaques on their cytoplasmic faces are composed of aligned ridges. We suggest that they are called ribbed adherens junctions. Perturbations of cell shaping and positioning indicate that loss o

关键词：黏着连接/病理学黏着连接/超微结构细胞大小细胞存活细胞骨架/病理学细胞骨架/超微结构聋/遗传学聋/病理学小鼠突变型显微镜检查电子动物小鼠

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Equine herpes virus type 1 (EHV-1) infection induces alterations in the cytoskeleton of Vero cells but not apoptosis

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ARCHIVES OF VIROLOGY 1999年第9期144卷 1827-1836页

作者： Walter, I Nowotny, N Univ Vet Med Inst Histol & Embryol A-1210 Vienna Austria Univ Vet Med Inst Virol A-1210 Vienna Austria

Effects of infection with two different strains of equine herpes virus type 1 (EHV-1;Fiber 178/83, Kentucky D) on the cytoskeleton of Vero cells were investigated immunohistochemically, and evaluated by confocal laser scanning microscopy. Twenty four hours post EHV-1 infection the assembly of the microtubulus system of Vero cells was heavily disturbed. The Golgi region was dispersed into vesicles spread throughout the cytoplasm as demonstrated by WGA lectin binding. Other cytoskeletal elements such as cytokeratin, vimentin, and filamentous actin (F-actin) were not affected by EHV-1 infection. The nature of Vero cell death after EHV-1 infection was investigated by three different methods to include all possible stages of apoptosis. All methods failed to demonstrate characteristic apoptotic features, therefore, it seems likely that necrosis is the predominant way of cell death in EHV-1 infected Vero cells.

关键词：细胞凋亡长尾猴黑细胞骨架/超微结构巨细胞/超微结构疱疹病毒1型马/生理学免疫组织化学外源凝集素类/代谢显微镜检查共焦坏死微管蛋白/代谢 Vero细胞动物小鼠

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Tyrosine phosphorylation and subcellular localization of focal adhesion proteins during in vitro decidualization of human endometrial stromal cells

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ENDOCRINOLOGY 1999年第12期140卷 5982-5990页

作者： Maruyama, T Yoshimura, Y Sabe, H Keio Univ Sch Med Dept Obstet & Gynecol Shinjuku Ku Tokyo 1600016 Japan Kyoto Univ Inst Virus Res Dept Biol Responses Kyoto 6068397 Japan

Human endometrial stromal cells undergo in vitro decidualization when treated with progesterone and estrogen. Using this model, we previously reported specific changes in the c-Src kinase activity and tyrosine phosphorylation of several proteins during in vitro decidualization, Focal adhesion kinase (FAK) and paxillin are known to form a complex with c-Src at the focal contacts and to participate in the integrin-mediated signal transduction as c-Src substrates. We here examined the tyrosine phosphorylation and subcellular localization of the focal adhesion proteins in stromal cells isolated from human endometrium. We found, however, that the total levels of FAK and paxillin tyrosine phosphorylation were not markedly changed during decidualization or after steroid withdrawal. In our culture system, numerous multicellular nodules were developed in cultures of decidualized stromal cells, within whose nodules the focal contacts were found to disappear. Moreover, disruption of the focal contacts was accompanied by disorganization of the actin-based cytoskeleton. These findings suggest that tyrosine phosphorylation of the endometrial paxillin and FBR is not tightly regulated by the kinase activity of c-Src during in vitro decidualization. The escape from regulation by c-Src may be in part due to the dissociation of the focal adhesion proteins/c-Src complex caused by the breakdown of the focal adhesion plaques as well as the loss of the actin-based cytoskeletal architecture.

关键词：细胞黏附分子/分析细胞黏附分子/代谢细胞支架蛋白质类/分析细胞支架蛋白质类/代谢细胞骨架/超微结构蜕膜/生理学子宫内膜/细胞学黏着斑激酶1 黏着斑蛋白酪氨酸激酶类桩蛋白磷蛋白类/分析磷蛋白类/代谢磷酰化磷酸酪氨酸/代谢蛋白酪氨酸激酶类/分析蛋白酪氨酸激酶类/代谢间质细胞/代谢间质细胞/超微结构亚细胞部分/化学女(雌)性人类

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Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons

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NEURON 1999年第2期23卷 247-256页

作者： Francis, F Koulakoff, A Boucher, D Chafey, P Schaar, B Vinet, MC Friocourt, G McDonnell, N Reiner, O Kahn, A McConnell, SK Berwald-Netter, Y Denoulet, P Chelly, J Inst Cochin Genet Mol INSERM U129 F-75014 Paris France Coll France CNRS UPR 9065 Biochim Cellulaire Lab F-75005 Paris France Stanford Univ Dept Biol Sci Stanford CA 94305 USA Weizmann Inst Sci Dept Mol Genet IL-76100 Rehovot Israel

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

关键词：抗体特异性脑/细胞学脑/胚胎学脑/代谢细胞分化/生理学细胞运动/生理学细胞培养的细胞骨架/代谢细胞骨架/超微结构免疫组织化学原位杂交微管相关蛋白质类/生物合成微管相关蛋白质类/生理学神经元/代谢神经元/生理学神经元/超微结构神经肽类/生物合成神经肽类/生理学磷蛋白类/生物合成磷蛋白类/生理学 RNA 信使/生物合成大鼠 Long-Evans 微管蛋白/分离和提纯微管蛋白/代谢动物小鼠大鼠

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Localisation of actin, villin, fimbrin, ezrin and ankyrin in rat taste receptor cells

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HISTOCHEMISTRY AND CELL BIOLOGY 1999年第1期112卷 79-86页

作者： Höfer, D Drenckhahn, D Univ Wurzburg Inst Anat D-97070 Wurzburg Germany

Mammalian taste buds consist of 50-150 pear- or spindle-shaped taste receptor cells which contain, at their apical cell surface, a bundle of microvillar projections. The microvilli probably serve to increase the receptive membrane surface of the chemosensory receptor cells. The molecular basis controlling the ultrastructure of taste receptor microvilli is present unknown. In the present study we analysed, by immunostaining at the light and electron microscopic levels and by immunoblotting, components of the cytoskeleton of these microvilli. We show here that taste cell microvilli contain the major cytoskeletal proteins of intestinal microvilli, actin, fimbrin and villin. Another actin-binding, peripheral membrane protein of intestinal microvilli, ezrin, was also localised to taste cell microvilli, where ezrin might play a role, for example, in placement of specific membrane proteins to the microvillus membrane. In search of further linkage proteins, we found ankyrin localised along the basolateral cell surface of taste receptor cells, where ankyrin might be involved in the immobilisation of the Na+,K+-ATPase or other ion-translocating proteins of taste cells to the membrane cytoskeleton.

关键词：肌动蛋白类/分析锚蛋白类/分析载体蛋白质类/分析化学感受器/化学化学感受器/超微结构细胞支架蛋白质类细胞骨架/化学细胞骨架/超微结构荧光抗体技术间接免疫印迹法免疫酶技术膜糖蛋白类/分析微丝蛋白质类/分析显微镜检查免疫电子磷蛋白类/分析大鼠 Wistar 味蕾/化学味蕾/超微结构动物女(雌)性男(雄)性大鼠

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Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization

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BLOOD 2000年第7期96卷 2487-2495页

作者： Mondoro, TH White, MM Jennings, LK Univ Tennessee Vasc Biol Program Memphis TN 38163 USA US FDA Ctr Biol Evaluat & Res Bethesda MD USA

Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction;however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADR addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa-mediated ligand binding can be separated mechanistically from GPIIb-IIIa-mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.(Blood. 2000;96:2487-2495) (C) 2000 by The American Society of Hematology.

关键词：腺苷二磷酸/药理学抗体/药理学结合部位血小板/生理学血小板/超微结构血块凝缩细胞骨架/代谢细胞骨架/超微结构纤维蛋白原/代谢碘放射性同位素显微镜检查电子扫描血小板聚集/药物作用血小板聚集抑制剂/药理学血小板糖蛋白GPⅡb-Ⅲa复合物/化学血小板糖蛋白GPⅡb-Ⅲa复合物/免疫学血小板糖蛋白GPⅡb-Ⅲa复合物/代谢蛋白质构象踝蛋白/代谢人类

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