The Roles of Cby1L and ciBAR1 in Spermatogenesis

项目摘要 精子鞭毛是一种经过改造的能动纤毛，对卵子的运动和受精至关重要。作为 这种鞭毛发生缺陷与不育有关。虽然不完全等同，但类似的机制 被认为在鞭毛和纤毛发生过程中起作用。纤毛发生缺陷与人类 这些疾病被称为纤毛病，如原发性纤毛运动障碍，并且许多患者还存在 生育问题。尽管大量的研究努力来了解纤毛蛋白在这些疾病中的作用， 它们在男性生育力中的生理作用仍不明确。我们报道了缺乏关键纤毛的小鼠 蛋白Chibby 1（Cby 1）显示经典纤毛病的迹象。分子上，Cby 1与细胞膜相互作用， 结合Bin/Amphiphysin/Rvs（BAR）结构域蛋白，Cby 1-相互作用的含BAR结构域1和2（ciBAR 1 和2，以前称为FAM 92 A和B），以促进纤毛发生。最近，我们发现奇比 家族成员，Cby 1-Like（Cby 1 L，也称为Cby 3），大量表达，几乎只在 睾丸我发现Cby 1 L和ciBAR 1定位于环状结构，这是鞭毛上基于隔蛋白的环状结构， 睾丸Cby 1 L-/-和ciBAR 1-/-雄性小鼠都是不育的，大多数精子显示出扭结的尾巴。在 此外，我发现Cby 1 L与ciBAR 1和环隔蛋白SEPT 4物理相互作用。有趣的是，在 Cby 1 L或ciBAR 1缺失，瓣环存在，但未能停止在中段/主段（MP/PP） 连接并向后进展到PP中。这些数据表明，Cby 1 L和ciBAR 1形成复合物， 有助于在MP/PP接合处正确定位瓣环。尽管环通常被约束到 膜在迁移过程中，在Cby 1 L-/-睾丸，环部分脱离鞭毛膜， 这表明Cby 1 L参与将纤维环锚定到膜上。总的来说，我的初步数据 提出了一个工作模型，其中Cby 1 L和ciBAR 1的复合物在准确的细胞内表达中起着不可或缺的作用。 在MP/PP接合处定位瓣环。本提案的总体目标是了解 Cby 1 L和ciBAR 1在精子发生和环定位中的分子作用。为了实现这一目标， 本研究的具体目的如下：1、明确Cby 1 L在鞭毛发生和鞭毛环迁移中的作用。我会 确定Cby 1 L在精子发生过程中的亚细胞定位，并研究其分子生物学特性。 Cby 1 L-/-小鼠雄性不育的细胞基础。2、研究Cby 1 L/ciBAR 1/septin相互作用， ciBAR 1在精子尾部分割中的作用。我预测Cby 1 L与ciBAR 1和环隔蛋白形成复合物 介导环的鞭毛膜的附着，这有助于适当的 精子尾部的分隔。同时，我将确定ciBAR 1-/- male中男性不育的原因。 小鼠总的来说，我希望我提出的研究将揭示一种新的机制， 环，由纤毛蛋白，这有助于适当的精子尾部分割。

Project Summary The sperm flagellum is a modified motile cilium that is essential for locomotion and fertilization of the egg. As such, defective flagellogenesis is linked to infertility. Although not completely equivalent, similar mechanisms are thought to operate during flagellogenesis and ciliogenesis. Defects in ciliogenesis are associated with human disorders, known as ciliopathies, such as primary ciliary dyskinesia, and many of the patients also present with fertility issues. Despite tremendous research efforts to understand the role of ciliary proteins in these diseases, their physiological roles in male fertility remain poorly defined. We reported that mice lacking the critical ciliary protein Chibby 1 (Cby1) display signs of classical ciliopathies. Molecularly, Cby1 interacts with the membrane binding Bin/Amphiphysin/Rvs (BAR) domain proteins, Cby1-interacting BAR domain-containing 1 and 2 (ciBAR1 and 2, formerly known as FAM92A and B), to facilitate ciliogenesis. More recently, we found that the Chibby family member, Cby1-Like (Cby1L, also known as Cby3), is expressed abundantly and almost exclusively in the testis. I found that Cby1L and ciBAR1 localize to the annulus, a septin-based ring structure on the flagellum, in the testis. Both Cby1L-/- and ciBAR1-/- male mice are infertile with the majority of sperm showing kinked tails. In addition, I found that Cby1L physically interacts with ciBAR1 and the annulus septin SEPT4. Interestingly, in the absence of Cby1L or ciBAR1, the annulus is present but fails to stop at the midpiece/principal piece (MP/PP) junction and progresses posteriorly into the PP. These data suggest that Cby1L and ciBAR1 form a complex that assists in proper positioning of the annulus at the MP/PP junction. Although the annulus is typically bound to membranes during migration, in Cby1L-/- testis, the annulus is partially detached from the flagellar membrane, suggesting that Cby1L is involved in anchoring the annulus to the membrane. Collectively, my preliminary data suggest a working model in which a complex of Cby1L and ciBAR1 plays an indispensable role in the accurate positioning of the annulus at the MP/PP junction. The overall goal of this proposal is to understand the molecular roles of Cby1L and ciBAR1 in spermatogenesis and annulus positioning. To achieve this goal, I propose the following Specific Aims: 1, Define the role of Cby1L in flagellogenesis and annulus migration. I will determine the subcellular localization of Cby1L during spermatogenesis and investigate the molecular and cellular basis of the male infertility of Cby1L-/- mice. 2, Investigate Cby1L/ciBAR1/septin interactions and ciBAR1’s role in sperm tail segmentation. I predict that Cby1L forms a complex with ciBAR1 and annulus septins to mediate the attachment of the annulus to the flagellar membrane, which contributes to the proper compartmentalization of the sperm tail. In parallel, I will determine the cause of male infertility in ciBAR1-/- male mice. Overall, I expect that my proposed research will unravel a novel mechanism for the proper positioning of the annulus, by ciliary proteins, which contributes to proper sperm tail segmentation.