Unique kinetics of Oct3/4 microlocalization following dissociation of human embryonic stem cell colonies
Unique kinetics of Oct3/4 microlocalization following dissociation of human embryonic stem cell colonies
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DOI：
10.1016/j.aanat.2012.05.004
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发表时间：
2013-01-01
影响因子：
2.2
通讯作者：
Sasaki, Katsunori
中科院分区：
文献类型：
--
作者：
Ichikawa, Hinako;Kanoh, Yoshiya;Sasaki, Katsunori
为了研究rho依赖性蛋白激酶（ROCK）抑制剂Y-27632对解离的人胚胎干（hES）细胞中E-cadherin、F-actin和ocl3 /4分布动力学的影响，并分析它们之间的相互作用形态学，在解离后24小时内对Y-27632处理的[R-i(+)]和未处理的[R-i(-)]细胞进行E-cadherin和ocl3 /4以及F-actin的免疫组织化学染色。实时荧光定量RT-PCR检测E-cadherin、Oct3/4、RhoA基因表达。E-cadherin的表达沿R-i（+）细胞膜或细胞簇的细胞间连接处呈线性增强。f -肌动蛋白沿细胞外周聚集，并沿细胞簇连接处呈网状扩张，Oct3/4在解离后数小时内被限制在细胞核内。然而，R-i（-）细胞表现出变形和起泡，并随着时间的推移而死亡。e -钙粘蛋白沿细胞外周呈点状分布，然后在细胞质的一侧或两侧聚集。肌动蛋白丝集中在泡底。在细胞质中检测到Oct3/4，而在细胞核中未检测到E-cadherin分布的恢复。实时定量RT-PCR结果显示，分离后12 h RhoA上调，E-cadherin下调。ROCK抑制未影响Oct3/4基因表达。这些结果表明，he细胞的协同性是由E-cadherin - actin细胞骨架系统维持的，同时Oct3/4在细胞核中的分布受到限制。RhoA激活后的解离使该系统紊乱，加速细胞死亡，而ROCK抑制部分抑制了这一过程。(C) 2012 Elsevier GmbH版权所有。
To investigate the effects of the Rho-dependent protein kinase (ROCK) inhibitor Y-27632 on the kinetics of E-cadherin, F-actin, and Oct3/4 distributions in dissociated human embryonic stem (hES) cells and to analyze their interactions morphologically, Y-27632-treated [R-i (+)] and untreated [R-i (-)] cells were immunohistochemically stained for E-cadherin and Oct3/4 within 24h of dissociation and also for F-actin. Furthermore, the gene expression of E-cadherin, Oct3/4, and RhoA was confirmed by quantitative real-time RT-PCR.E-cadherin expression intensified linearly along the membranes of R-i (+) cells or intercellular junctions in cell clusters. F-actin accumulated along the periphery of cells and expanded in a web-like manner along junctions in cell clusters, and Oct3/4 was restricted to the nucleus within few hours of dissociation. However, R-i (-) cells exhibited deformation and blebbing and appeared to die over time. E-cadherin exhibited a punctate pattern along the periphery, after which it accumulated on one or both sides of the cytoplasm. Actin filaments were concentrated at the bleb bases. Oct3/4 was detected in the cytoplasm, not in the nucleus the recovery of integrated E-cadherin distribution. Quantitative real-time RT-PCR revealed RhoA upregulation and E-cadherin downregulation at 12 h after dissociation. Oct3/4 gene expression was unaffected by ROCK inhibition.These results revealed that the cooperative nature of hES cells is maintained by the E-cadherin actin cytoskeleton system along with the restricted distribution of Oct3/4 in the nucleus. RhoA activation followed by dissociation disorders this system and accelerates cell death, which is partially suppressed by ROCK inhibition. (C) 2012 Elsevier GmbH. All rights reserved.