上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

FRAP技术揭示PLZF-RARα融合蛋白在细胞核内的运动性依赖于其配基及转录活性

贺 薇，黄 莺   

  1. 上海交通大学  基础医学院病理生理学教研室  细胞分化与凋亡教育部重点实验室， 上海 200025
  • 出版日期:2014-10-28 发布日期:2014-10-28
  • 作者简介:贺 薇（1987—）， 女， 硕士生; 电子信箱: hewei_0718@126.com。

Dependency of intranuclear mobility of PLZF-RARα fusion proteins on ligand and its transcription activity revealed by FRAP

HE Wei, HUANG Ying   

  1. Department of Pathophysiology, the Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Basic Medicine Faculty of Shanghai Jiao Tong University, Shanghai 200025, China
  • Online:2014-10-28 Published:2014-10-28

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摘要：

目的 运用光漂白荧光恢复（FRAP）技术检测一系列带有绿色荧光蛋白（GFP）标签的野生型及突变型的早幼粒细胞性白血病锌指-维甲酸受体α（PLZF-RARα）表达蛋白分子在细胞核内运动性。方法 运用凝胶迁移实验（EMSA）检测带有GFP标签的野生型及突变型PLZF-RARα融合蛋白结合维甲酸反应元件（RARE）的能力；运用FRAP以及共聚焦荧光显微镜分析一系列GFP-PLZF-RARα及其突变体表达蛋白在细胞核内的运动性；运用转录抑制剂放线菌素D （Act D）和/或全反式维甲酸（ATRA）处理GFP-RARα和GFP-PLZF-RARα表达细胞，运用FRAP观察PLZF-RARα和野生型RARα在细胞核内的运动性变化情况。结果 位于PLZF结构区的POZ以及锌指结构域不仅是引起PLZF-RARα核内运动性降低的主要原因，同时也是引起配体诱导的核内运动性减慢的关键因素。Act D能够显著地抑制PLZF-RARα和RARα蛋白分子在细胞核内的运动性；分化诱导剂ATRA处理可逆转Act D引起的RARα核内运动性降低效应，但经Act D处理的PLZF-RARα分子无此效应。结论 运用FRAP技术发现PLZF-RARα融合蛋白在细胞核内的运动性减慢可能依赖于配体的存在及其转录活性。

关键词: 早幼粒细胞性白血病锌指-维甲酸受体α, 维甲酸受体α, 绿色荧光蛋白, 光漂白荧光恢复, 转录

Abstract:

Objective To detect the mobility of a series of green fluorescence protein (GFP) tagged wild-type and mutant promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) expressing proteins by fluorescence recovery after photobleaching (FRAP). Methods The electrophoretic mobility shift assay (EMSA) was adopted to detect the ability of GFP tagged wild-type and mutant PLZF-RARα fusion protein binding with retinoic acid response elements (RARE). The intranuclear mobility of PLZF-RARα and its mutant expressing proteins was analyzed by FRAP and confocal fluorescence microscopy. GFP-RARα and GFP-PLZF-RARα expressing cells were treated by transcriptional inhibitor actinomycin D (Act D) and/or ATRA. Variations of the intranuclear mobility of PLZF-RARα and wild-type RARα under these treatments was observed by FRAP. Results POZ and zinc finger domains within PLZF moiety not only contributed to the reduction of intranuclear mobility of PLZF-RARα, but also caused the decrease of ligand-induced intranuclear mobility. Act D could effectively immobilize the intranuclear mobility of PLZFRARα and RARα protein molecules. Treatment with differentiation inducer all-trans retinoic acid (ATRA) could reverse Act D induced immobilization of the intranuclear mobility of RARα, but could not reverse the immobilization of intranuclear mobility of PLZF-RARα treated by Act D. Conclusion Decreased intranuclear mobility of PLZF-RARα fusion protein detected by FRAP may rely on the existence of ligand and its transcription activity.

Key words: promyelocytic leukemia zinc finger-retinoic acid receptor α, retinoic acid receptor α, green fluorescence protein, fluorescence recovery after photobleaching, transcription