Robertson Claire, Church Stewart W, Nagar Hans A, Price John, Hall Peter A, Russell S E Hilary
Department of Oncology, Queen's University Belfast, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland, UK.
J Pathol. 2004 May;203(1):519-27. doi: 10.1002/path.1551.
Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions. Alterations in the levels of Sept9 protein by overexpression of individual isoforms can clearly perturb cellular behaviour and may thus provide a mechanistic explanation for observations of deranged septin expression in neoplasia.
进化上保守的septin基因家族成员正逐渐成为包括膜运输、胞质分裂和细胞周期控制事件在内的多种细胞过程的关键组成部分。SEPT9已被证明具有复杂的基因组结构，通过五个可变氨基末端和三个可变羧基末端的重排，可能产生多达15种不同的异构体。SEPT9的基因组和转录改变与肿瘤形成有关。本研究使用一种Sept9特异性抗体来确定一系列肿瘤细胞系中异构体的表达模式。蛋白质印迹分析表明Sept9的相对含量和异构体含量存在显著差异。免疫荧光研究显示了一系列细胞质定位模式，从主要为颗粒状到主要为丝状。还针对每个氨基末端异构体构建了表达载体，以研究单个异构体的定位模式以及异位表达对细胞的影响。本研究表明，在这种过表达系统中，ε异构体呈丝状，而其余异构体为颗粒状且位于细胞质中。将单个构建体瞬时转染到肿瘤细胞系中会导致细胞周期紊乱，出现G2/M期阻滞并显著抑制生长，这在Sept9内源性含量最低的细胞系中最为明显。对Sept9所有五个N末端变体的GTP结合突变体也进行了类似的表型观察。然而，在转染细胞中野生型与突变型septin蛋白积累的动力学方面观察到了显著差异。总之，本研究表明Sept9蛋白的表达模式在一组肿瘤细胞系中非常多样，功能研究与septin作为分子支架的组成部分发挥作用的模型一致，该分子支架有助于多种细胞功能。通过单个异构体的过表达改变Sept9蛋白水平可明显扰乱细胞行为，因此可能为肿瘤中septin表达紊乱的观察结果提供一种机制解释。
