Vaid Kuljeet S, Guttman Julian A, Singaraja Roshni R, Vogl A Wayne
Department of Cellular and Physiological Sciences, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
Biol Reprod. 2007 Dec;77(6):1037-48. doi: 10.1095/biolreprod.107.063735. Epub 2007 Sep 12.
During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.
在精子发生过程中，精子细胞在生精上皮中经历“上下”易位事件。有人提出，这一事件是由外质特化结构的移动导致的，外质特化结构在支持细胞与精子细胞的黏附位点形成，并沿着相邻的微管束移动。为了验证驱动蛋白与外质特化结构相关的假说，我们针对保守的驱动蛋白序列制备了抗体，并在固定的冷冻睾丸切片和固定的生精上皮片段上检测驱动蛋白。这些抗体除了与上皮中已知含有驱动蛋白的其他结构发生反应外，还与精子细胞相关的外质特化结构发生反应。在电子显微镜水平上，这些抗体与外质特化结构的内质网成分的胞质面发生反应。基于使用小鼠睾丸和支持细胞基因芯片阵列进行的mRNA转录本筛选，我们确定KIF20是外质特化结构处的一种候选驱动蛋白。针对这种驱动蛋白特有的肽序列制备的抗体，在睾丸切片和上皮片段的外质特化结构处发生反应，并且在通过电子显微镜分析时，也与外质特化结构的内质网成分发生反应。该抗体在蛋白质免疫印迹上与全长KIF20发生反应。在睾丸裂解物的蛋白质免疫印迹上，该抗体与一种其他组织中不存在的蛋白质发生反应，该蛋白质的迁移分子量高于KIF20的预测分子量。我们的结果表明，一种驱动蛋白与支持细胞顶端的外质特化结构相关，并且这种分子马达可能是KIF20的一种同工型。
