EBP50影响HeLa细胞微丝骨架的分布和定位

EBP50 Influences Distribution and Localization of Microfilament Cytoskeleton in Cultured HeLa Cells

目的:通过研究与膜-细胞骨架连接蛋白(ezrin-radixin-moesin,ERM)家族相结合的磷酸化蛋白50(ERM-binding phospho-protein-50,EBP50)对HeLa细胞微丝骨架含量、分布的影响及受血小板源性生长因子PDGF(platelet-derived growth factor)刺激后,微丝骨架在细胞中定位的变化及微丝骨架定位变化与EBP50的关系,阐明EBP50蛋白影响肿瘤细胞生长迁移的分子机制。方法:将pBK-CMV-HA空载体和pBK-CMV-HA-EBP50wt重组质粒分别转染HeLa细胞系,G418进行稳定表达细胞系的筛选,并用Western免疫印迹方法进行蛋白表达的鉴定;利用Western免疫印迹、免疫荧光细胞化学染色等方法结合光镜和激光共聚焦扫描显微镜分别观察和分析HA-HeLa与HA-EBP50-HeLa微丝骨架的含量及分布的异同;然后使用10ng/ml和20ng/mlPDGF37℃刺激细胞15min后,分别观察及分析两组细胞微丝骨架的分布情况及EBP50在细胞中定位的变化。结果:Western免疫印迹鉴定证实转染的外源性EBP50cDNA片段可在HeLa细胞系中成功表达EBP50蛋白,证明获得稳定表达EBP50蛋白的HeLa细胞系。Western免疫印迹及免疫荧光结果证实,转染空载pBK-CMV-HA的HeLa细胞微丝骨架粗大疏松,方向不一,交错排列;与其相比,EBP50虽然对HeLa细胞微丝骨架的含量没有明显影响,但能够促使细胞微丝骨架呈致密细丝状,平行规则排列,并沿细胞极性分布;并且在PDGF刺激下,EBP50能够与微丝骨架一起自胞浆迁移至膜上,并共定位。结论:EBP50能改变HeLa细胞微丝骨架的分布。在受到PDGF刺激时,EBP50还能使HeLa细胞系的微丝骨架定位于膜表面。EPB50可能通过影响微丝骨架的分布和定位来发挥其影响肿瘤细胞生长迁移的功能。

Objective： To explore the influence of EBP50 （ezrin-radixin-moesin-binding phospho-protein-50） on microfilament cytoskeleton content and distribution in cultured HeLa cells, to investigate the relationship between the changes in microfilament cytoskeleton localization and EBP50 after PDGF （platelet-derived growth factor） stimulation, and to further clarify the molecular mechanism by which EBP50 suppresses tumor cell proliferation and migration..Methods： pBK-CMV-HA-EBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were transfected into HeLa cells. G418 at 350mg/L was used to screen for cell clones stably expressing EBP50. Western blot was carried out to detect EBP50 expression. Similarities and differences in microfilament cytoskeleton content and distribution in HeLa cells transfected with pBK-CMV-HA-EBP50 wild type recombinant plasmid or pBK-CMV-HA empty vector were analyzed by Western blot, fluorescence staining and confocal microscopy. HeLa cells stably transfected with the pBK-CMV-HA-EBP50 wild type recombinant plasmid and the pBK-CMV-HA empty vector were also treated with PDGF （10 ng/ml and 20 ng/ml, 37℃, 15min） and stained by rhodamine-labeled phalloidin to observe the distribution of microfilament cytoskeleton in the two groups. EBP50 protein distribution in PDGF-stimulated HeLa cells was detected by immunofluorescence. Results： Western blot results confirmed that the EBP50 cDNA fragment could express EBP50 in cul- tured HeLa cell lines and that cell lines stably expressing EBP50 were successfully obtained. Western blot and fluorescence results showed that in the cell line transfected with empty vector, the microfilament cytoskeleton was thick, loose, multidirectional and displayed crossing arrangements. The content of microfilamerit cytoskeleton in the cell line transfected with pBK-CMV-HA-EBP50 was different from that found in the cell line transfected with empty vector. EBP50 expression enhanced microfilament cytoskeleton polymerization into compact thin filaments. Under the stimulation of PDGF, EBP50 migrated to the cell membrane from the cytosol together with microfilament cytoskeleton and co-localized there. Conclusion： EBP50 can change the distribution of microfilament cytoskeleton in cultured HeLa cells and can also bind the microfilament cytoskeleton to the cell membrane under the stimulation of PDGF. EBP50 may play a role in the proliferation and migration of tumor cells by influencing the distribution and localization of microfilament cytoskeleton.