Pu Jiali, Li Yingzhi, Liu Zhirong, Yan Yaping, Tian Jun, Chen Sheng, Zhang Baorong
Department of Neurology (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Mol Vis. 2011 Feb 24;17:591-7.
FERM domain containing 7 (FRMD7) is a member of the four-point-one, ezrin, radixin, moesin (FERM) family of proteins, and has been reported to cause X-linked idiopathic congenital nystagmus (ICN), a disease which affects ocular motor control. There have been over 30 mutations reported for FRMD7, however, their role in the pathogenesis of ICN remains unclear. The purpose of this study is to perform the expression distributes of protein FRMD7 from human fetal brain during development and to understand the relationship with cytoskeletal protein F-actin between wild-type and mutation-type FRMD7.
Expression of protein FRMD7 from developing human fetal brain was tested by immunohistochemistry. Enhanced green fluorescent protein (EGFP)-tagged recombinant plasmids DNA encoding the normal or mutant FRMD7 were used to transiently transfect the mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). Further, confocal microscopic analysis was used to determine the subcellular localization of the fusion proteins. To visualize F-actin, fixed HEK293T cells were stained with rhodamine-phalloidin.
We show that expression of FRMD7 was mainly detected in the brainstem (a region associated with ocular motor control), while limited level was observed in the cortex. The COOH-terminus of FRMD7 was found to play a key role in the subcellular localization of FRMD7 in mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). While no differences in the co-localization of F-actin between the wild-type and missense mutation-type (c.781C>G and c.886G>C) proteins was observed, an additional mutant, c.1003C>T, which results in a COOH-terminally truncated protein, exhibited a nuclear localization pattern which did not co-localize with the cytoplasmic distribution of F-actin.
The results of the present study indicate that FRMD7 may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics.
含FERM结构域蛋白7（FRMD7）是四点-蛋白、埃兹蛋白、根蛋白、膜突蛋白（FERM）家族的成员，据报道可导致X连锁特发性先天性眼球震颤（ICN），这是一种影响眼球运动控制的疾病。已报道FRMD7有30多种突变，然而，它们在ICN发病机制中的作用仍不清楚。本研究的目的是检测发育过程中人类胎儿大脑中FRMD7蛋白的表达分布，并了解野生型和突变型FRMD7与细胞骨架蛋白F-肌动蛋白之间的关系。
通过免疫组织化学检测发育中的人类胎儿大脑中FRMD7蛋白的表达。使用编码正常或突变型FRMD7的增强型绿色荧光蛋白（EGFP）标记的重组质粒DNA瞬时转染小鼠神经母细胞瘤细胞（Neuro-2a）和人胚肾293细胞（HEK293T）。此外，共聚焦显微镜分析用于确定融合蛋白的亚细胞定位。为了可视化F-肌动蛋白，用罗丹明-鬼笔环肽对固定的HEK293T细胞进行染色。
我们发现FRMD7的表达主要在脑干（与眼球运动控制相关的区域）检测到，而在皮质中观察到的水平有限。发现FRMD7的COOH末端在小鼠神经母细胞瘤细胞（Neuro-2a）和人胚肾293细胞（HEK293T）中FRMD7的亚细胞定位中起关键作用。虽然野生型和错义突变型（c.781C>G和c.886G>C）蛋白之间F-肌动蛋白的共定位没有差异，但另一个突变体c.1003C>T导致COOH末端截短的蛋白，表现出核定位模式，与F-肌动蛋白的细胞质分布不共定位。
本研究结果表明，FRMD7可能在人类胎儿大脑发育早期的脑干中起重要作用，并为FRMD7突变的机制提供了线索，该机制可能参与影响F-肌动蛋白动力学。
