研究者詳細 - 植木　尚子

A cell-to-cell macromolecular transport assay in Planta utilizing biolistic bombardment. 査読

Ueki S, Meyers BL, Yasmin F, Citovsky V

Journal of visualized experiments : JoVE ( 42 ) 2010年8月

Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment 査読

Shoko Ueki, Benoit Lacroix, Alexander Krichevsky, Sondra G. Lazarowitz, Vitaly Citovsky

NATURE PROTOCOLS 4 ( 1 ) 71 - 77 2009年

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Transient gene expression is an indispensable tool for studying functions of gene products. In the case of plants, transient introduction of genes by Agrobacterium infiltration is a method of choice for many species. However, this technique does not work efficiently in Arabidopsis leaf tissue, the most widely used model system for basic plant biology research. Here we present an optimized protocol for biolistic delivery of plasmid DNA into the epidermis of Arabidopsis leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol yields efficient and reproducible transient expression of diverse genes and is exemplified here for use in a functional assay of a transcription repressor and for the subcellular localization and cell-to-cell movement of plant viral movement protein. This protocol is suitable for studies of biological function and subcellular localization of the gene product of interest directly in planta by utilizing different types of activity-based assays. Using this procedure, the data are obtained after 2-4 d of work.

DOI： 10.1038/nprot.2008.217

Identification of an interactor of cadmium ion-induced glycine-rich protein involved in regulation of callose levels in plant vasculature 査読

S Ueki, Citovsky, V

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 ( 34 ) 12089 - 12094 2005年8月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

Cadmium-induced glycine-rich protein (cdiGRP) is a cell wall-associated factor that increases callose levels in plant vasculature. To better understand the cdiGRP/callose regulation system, we identified a tobacco protein, GrIP (cdiGRP-interacting protein, GrIP), that associates with cdiGRP and localizes at the plant cell wall. Constitutive overexpression of GrIP enhanced the accumulation of the cdiGRP protein and callose in vasculature-associated cells with or without treatment with cadmium ions. That GrIP gene expression was not affected by cadmium ions indicated that GrIP does not directly modulate the callose levels induced by the treatment. Instead, GrIP most likely functions by further elevating the accumulated amount of cdiGRP, the expression of which is up-regulated by the cadmium ions. Interestingly, the levels of cdiGRP mRNA were not affected by constitutive expression of GrIP, demonstrating that the enhancement in cdiGRP protein accumulation by GrIP overexpression occurs posttranslationally. Collectively, these observations suggest that GrIP interacts with cdiGRP and increases its level of accumulation; in turn, the elevated amounts of cdiGRP induce callose deposits in the plant cell walls. Therefore, GrIP and cdiGRP represent sequentially acting factors in a biochemical pathway that regulates callose accumulation in the plant vasculature.

DOI： 10.1073/pnas.0505927102

Control improves with age: Intercellular transport in plant embryos and adults 査読

S Ueki, Citovsky, V

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 ( 6 ) 1817 - 1818 2005年2月

The systemic movement of a tobamovirus is inhibited by a cadmium-ion-induced glycine-rich protein 査読

S Ueki, Citovsky, V

NATURE CELL BIOLOGY 4 ( 7 ) 478 - 485 2002年7月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Systemic movement is central to plant viral infection. Exposure of tobacco plants to low levels of cadmium ions blocks the systemic spread of turnip vein-clearing tobamovirus (TVCV). We identified a tobacco glycine-rich protein, cdiGRP, specifically induced by low concentrations of cadmium and expressed in the cell walls of plant vascular tissues. Constitutive cdiGRP expression inhibited systemic transport of TVCV, whereas suppression of cdiGRP production allowed TVCV movement in the presence of cadmium. cdiGRP exerted its inhibitory effect on TVCV transport by enhancing callose deposits in the vasculature. So cdiGRP may function to control plant viral systemic movement.

DOI： 10.1038/ncb806

RNA commutes to work: regulation of plant gene expression by systemically transported RNA molecules 査読

S Ueki, Citovsky, V

BIOESSAYS 23 ( 12 ) 1087 - 1090 2001年12月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COMPANY OF BIOLOGISTS LTD

Although long-distance movement of endogenous mRNAs in plants is well established, the functional contributions of these transported RNA molecules has remained unclear. In a recent report, Kim et al.((1)) showed that systemically transported mRNA is capable of causing phenotypic change In developing tissue. Here, this finding and its significance are reviewed and discussed In detail. In addition, in order to give proper perspective, long-distance transport of other types of RNAs, e.g., RNA elicitors of post-transcriptional gene silencing and RNA genomes of plant viruses, and its possible regulation are discussed. (C) 2001 John Wiley & Sons, Inc.

DOI： 10.1002/bies.10027

Inhibition of systemic onset of post-transcriptional gene silencing by non-toxic concentrations of cadmium 査読

S Ueki, Citovsky, V

PLANT JOURNAL 28 ( 3 ) 283 - 291 2001年11月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BLACKWELL SCIENCE LTD

Post-transcriptional gene silencing (PTGS) is an important mechanism for regulation of plant gene expression and virus-plant interactions. To better understand this process, the heavy metal cadmium was identified as a specific inhibitor in two different PTGS systems, constitutive and inducible. The pattern of cadmium-induced inhibition of PTGS allowed several insights into PTGS development. First, cadmium treatment prevented only systemic but not local onset of PTGS, uncoupling between these two modes of PTGS. Second, non-toxic, but not toxic, levels of cadmium inhibited PTGS, suggesting induction of a pathway that interferes with PTGS. Third, cadmium effects on PTGS closely paralleled those on the movement of tobamoviruses, suggesting that both processes may share common steps in their systemic transport pathways. Interestingly, these effects of cadmium do not represent a general property of toxic metal ions because two other such elements, that is zinc and aluminum, did not interfere with PTGS and viral systemic movement.

DOI： 10.1046/j.1365-313X.2001.01145.x

Activity-independent cell adhesion to tissue-type transglutaminase is mediated by alpha 4 beta 1 integrin 査読

T Isobe, H Takahashi, S Ueki, J Takagi, Y Saito

EUROPEAN JOURNAL OF CELL BIOLOGY 78 ( 12 ) 876 - 883 1999年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：URBAN & FISCHER VERLAG

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. We have previously reported that one of the TGases, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. In this paper, we report for the first time that tissue-type transglutaminase (TGc) also has cell adhesion activity TGc-coated plastic surface promoted adhesion and spreading of cells in a TGc concentration-dependent manner. However, there are some obvious differences between cell adhesion mediated by TGc and FXIIIa, As was reported previously, the adhesion to FXIIIa is dependent on its TGase activity In contrast, the TGc-mediated cell adhesion is independent of its TGase activity: 1) The modification of the active center cysteine with iodoacetamide blocked the enzyme activity without any effect on cell adhesion; 2) the addition of Mg2+ did not induce the enzyme activity, but it was as effective as Ca2+ for cell adhesion; 3) the addition of NH4+ inhibited the enzyme activity but did not affect the cell adhesion significantly. The integrins involved in these cell adhesions are quite different. In the case of FXIIIa, av()3 and alpha 5 beta 1 integrins are involved and consequently the RGD peptide substantially inhibited the adhesion. On the other hand, the cell adhesion to TGc is mediated by alpha 4 beta 1 integrin but not alpha 5 beta 1; a CS-1 peptide, which represents the binding site of fibronectin to alpha 4 beta 1 integrin, completely inhibited the cell adhesion to TGc, It is possible that TGc and FXIIIa mag mediate cell adhesion under different physiological and pathological situations.

12-hydroxy-5Z,8Z,10E,14Z, eicosatetraenoic acid (12-HETE) stimulates cAMP production in normal human fibroblasts 査読

S Ueki, J Takagi, Y Kobayashi, F Sato, Y Saito

JOURNAL OF CELLULAR PHYSIOLOGY 178 ( 1 ) 63 - 68 1999年1月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

We report here that the 12-lipoxygenase metabolite of arachidonic acid, 12-hydroxy-5Z, 8Z, 10E, 14Z, eicosatetraenoic acid (12-HETE), stimulates cAMP production in human fibroblasts among various cultured cell lines tested. Although 12-HETE seemed to stimulate the phospholipase C (PLC)-protein kinase C (PKC) system, inhibitors against PLC and PKC did not reduce the cAMP production induced by 12-HETE, indicating that the activation of PLC-PKC system is not positively coupled with the stimulation of cAMP production. On the other hand, the cAMP production induced by 12-HETE was dependent on the Ca2+/calmodulin system in the cells. The results suggest that 12-HETE specifically stimulates Ca2+/calmodulin-dependent adenylyl cyclase to increase cAMP level in the fibroblasts. (C) 1999 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-4652(199901)178:1<63::AID-JCP8>3.0.CO;2-J

The 'ligand-induced conformational change' of alpha 5 beta 1 integrin - Relocation of alpha 5 subunit to uncover the beta 1 stalk region 査読

J Tsuchida, S Ueki, Y Takada, Y Saito, J Takagi

JOURNAL OF CELL SCIENCE 111 1759 - 1766 1998年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COMPANY OF BIOLOGISTS LTD

Integrin heterodimers undergo a conformational change upon the binding of ligand to their extracellular domains. An anti-beta 1 integrin monoclonal antibody AG89 can detect such a conformational change since it recognizes a ligand-inducible epitope in the stalk-like region of beta 1 subunits, The binding of a I-125-labeled AG89 Fab fragment to alpha 5 beta 1 integrins on K562 cells was assessed and analyzed by the Scatchard method. High affinity binding sites for AG89 are present on cells treated with ligand peptide. In addition, results revealed that cells treated with EDTA also express AG89 binding sites with the same affinity although the number of binding sites is 4-fold lower. AG89 immunoprecipitated alpha 5 beta 1 complexes from surface-labeled K562 cells treated with ligand peptide. By contrast, it immunoprecipitated only beta 1 chains when the ligand peptide was absent, suggesting that high affinity binding sites on EDTA-treated cells are associated with nonfunctional beta 1 monomer. Additional studies show that the epitope for AG89 is constitutively exposed on mutant pi that cannot complex with alpha 5. These data suggest that the AG89 epitope is masked by the alpha 5 subunit. Ligand binding and integrin activation may uncover the beta 1 stalk region by triggering a conformational shift of alpha 5 relative to beta 1.

Classification of 'activation' antibodies against integrin beta 1 chain 査読

J Tsuchida, S Ueki, Y Saito, J Takagi

FEBS LETTERS 416 ( 2 ) 212 - 216 1997年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

We compared the effects of two anti-beta 1 integrin activating antibodies, TS2/16 and AG89, on K562 cell adhesion to fibronectin. Though both antibodies effectively induced cell adhesion, the EC50 for AG89 was more than 200-fold higher than that for TS2/16, Scatchard analysis of the data from [I-125]Fab fragment binding to the cells revealed that the TS2/16 epitope is exposed constitutively on all the beta 1 integrin molecules, while only 3% of the beta 1 integrins on resting K562 cells bear the AG89 epitope. Calculation of the actual number of each antibody bound to the cell during the cell adhesion assay revealed that induction of cell adhesion can be accomplished by binding much fewer AG89 molecules compared to TS2/16. Thus, AG89 and TS2/16 represent distinct classes of anti-integrin activating antibodies that show completely different binding characteristics as well as different activation effects on the integrin molecule upon binding. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)01206-4

Dual functions of transglutaminase in novel cell adhesion 査読

S Ueki, J Takagi, Y Saito

JOURNAL OF CELL SCIENCE 109 2727 - 2735 1996年11月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COMPANY OF BIOLOGISTS LTD

Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins, In the present study, we report for the first time that a representative enzyme, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells, When coated on plastic surfaces FXIIIa promoted adhesion and spreading of various cells of both normal and tumor origin, in a concentration-dependent manner, The adhesion was not inhibited by antibodies against possible contaminants in the enzyme preparation such as fibronectin and vitronectin, but was completely inhibited by a polyclonal antibody against the enzyme, Therefore, if there were any contaminating cell adhesive substrates in the enzyme preparation, they cannot account for the observed cell adhesion to the enzyme; FXIIIa itself mediates the cell adhesion, Furthermore, phosphorylation of tyrosine residues in 120 kDa and 70 kDa proteins was clearly shown in human fibroblasts adhering to the enzyme, Formation of actin stress fibers was also unambiguously observed in the adhering cells, These biochemical reactions, which are also observed when cells adhere to a typical cell adhesion protein, fibronectin, are believed to be of importance in the process of cell adhesion, This adhesion activity of FXIIIa was dependent on its TGase activity, because both a modification of the active center cysteine with iodoacetamide and the addition of ammonium ion abolished the cell adhesion activity along with the enzyme activity, The cell adhesion to fibronectin, however, was not affected by these treatments, The effects of various anti-integrin antibodies suggested that both alpha v beta 3 and beta 1 family integrins participated in the cell adhesion to FXIIIa, Taken together, these data demonstrate for the first time that there is a unique TGase activity-mediated cell adhesion, This novel function of the enzyme may be of physiological importance.