Redenbach D M, Boekelheide K, Vogl A W
Department of Anatomy, University of British Columbia, Vancouver, Canada.
Eur J Cell Biol. 1992 Dec;59(2):433-48.
Ectoplasmic specializations (ESs) are submembrane specializations that consist of Sertoli cell plasma membrane linked by an ordered array of actin filaments to a cisterna of endoplasmic recticulum (ESER). They are thought to function in the spermatid-Sertoli cell adhesion junction. Microtubules occur adjacent to the cytoplasmic face of the ESER and are oriented parallel to the long axis of the Sertoli cell, the direction of spermatid translocation during spermatogenesis. Our hypothesis that spermatid orientation and translocation in the seminiferous epithelium is microtubule dependent predicts that microtubules bind to ESs. To test for binding between microtubules and ESs, we have developed an in vitro assay in which spermatid-ES complexes were isolated from the seminiferous epithelium and incubated with bovine brain microtubules that were labeled with [3H]GTP and stabilized with taxol. Binding was determined by scintillation counts from gradient fractions enriched for spermatid-ES complexes and depleted of unbound microtubules by differential centrifugation. Our data indicate that microtubules bind to spermatid-ES complexes in a substrate concentration-dependent manner and can be released with 5 mM GTP or 10 mM MgATP. Binding is competitively reduced with excess unlabeled microtubules and is inhibited by 100 microM vanadate and 2 mM N-ethylmaleimide (NEM). The amount of binding is unchanged by 10 microM vanadate, 2 mM erythro-(2-hydroxy-3-nonyl)adenine (EHNA) or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP). Immunofluorescence and autoradiographic data confirm that labeled microtubules bind to ES locations on spermatid-ES complexes. These data are consistent with the hypothesis that spermatid translocation is a microtubule-based transport event.
胞质特化结构（ESs）是一种膜下特化结构，由支持细胞的质膜组成，通过肌动蛋白丝的有序排列与内质网池（ESER）相连。它们被认为在精子细胞与支持细胞的黏附连接中发挥作用。微管出现在ESER的细胞质面附近，并与支持细胞的长轴平行排列，这也是精子发生过程中精子细胞移位的方向。我们的假设是，生精上皮中精子细胞的定向和移位依赖于微管，这预测微管会与ESs结合。为了测试微管与ESs之间的结合，我们开发了一种体外试验，从生精上皮中分离出精子细胞-ES复合物，并与用[3H]GTP标记并用紫杉醇稳定的牛脑微管一起孵育。通过对富含精子细胞-ES复合物且通过差速离心去除未结合微管的梯度级分进行闪烁计数来确定结合情况。我们的数据表明，微管以底物浓度依赖的方式与精子细胞-ES复合物结合，并且可以用5 mM GTP或10 mM MgATP释放。过量未标记的微管会竞争性降低结合，并且100 microM钒酸盐和2 mM N-乙基马来酰亚胺（NEM）会抑制结合。10 microM钒酸盐、2 mM赤藓醇-（2-羟基-3-壬基）腺嘌呤（EHNA）或1 mM 5'-腺苷酰亚胺二磷酸（AMP-PNP）不会改变结合量。免疫荧光和放射自显影数据证实，标记的微管与精子细胞-ES复合物上的ES位置结合。这些数据与精子细胞移位是基于微管的运输事件这一假设一致。
