Subcellular Localization of Tektin2 in Rat Sperm Flagellum
Subcellular Localization of Tektin2 in Rat Sperm Flagellum
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DOI：
10.2108/zsj.27.755
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发表时间：
2010-09-01
影响因子：
0.9
通讯作者：
Iida, Hiroshi
中科院分区：
文献类型：
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作者：
Shimasaki, Sayaka;Yamamoto, Etsuko;Iida, Hiroshi
Tektins是进化上保守的细丝形成蛋白，定位于鞭毛和纤毛，据报道与轴丝微管的稳定性和结构复杂性有关。已经报道了五种哺乳动物Tektins(Tektin1-5)。其中，Tektin2(TEKT2)是正常鞭毛结构和功能所必需的。Tekt2缺失的精子表现出鞭毛弯曲和活力降低，可能是由于动力蛋白内臂的破坏。然而，TEKT2在精子中的亚细胞定位在超微结构水平上尚不清楚。为了阐明TEKT2在大鼠精子鞭毛中的分子定位，我们进行了激光共聚焦扫描显微镜、鞭毛提取和免疫印迹分析以及免疫金电子显微镜观察。用十二烷基硫酸钠-乙二胺四乙酸乙酯提取精子鞭毛，可以完全提取出轴丝微管蛋白，而TEKT2只从鞭毛中部分释放出来，提示TEKT2可能存在于轴毛周围成分中，与轴丝微管蛋白没有直接联系。激光共聚焦扫描显微镜和包埋前免疫电子显微镜显示，TEKT2与外密纤维(ODF)的表面有关。TEKT2可能是一种与ODF相关的分子，是鞭毛稳定和精子运动所必需的。
Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1-5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immuno-gold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.