The Roles of Cby1L and ciBAR1 in Spermatogenesis

项目摘要 精子鞭毛是一种改良的活动纤毛，对卵子的运动和受精是必不可少的。AS 这种有缺陷的鞭毛发生与不孕症有关。虽然不是完全等价，但类似的机制是 被认为在鞭毛发生和纤毛发生过程中起作用。纤毛发生缺陷与人类有关 被称为纤毛疾病的疾病，如原发性睫状体运动障碍，许多患者还表现为 生育问题。尽管在了解纤毛蛋白在这些疾病中的作用方面付出了巨大的研究努力， 它们在男性生育中的生理作用仍然没有明确的定义。我们报告了缺乏关键纤毛的小鼠 蛋白CHIBBY 1(Cby1)表现出典型的纤毛病变征象。在分子水平上，Cby1与膜相互作用 结合Bin/双体蛋白/RVS(BAR)结构域蛋白，Cby1-相互作用的BAR结构域包含1和2(ciBAR1 和2，以前称为FAM92A和B)，以促进纤毛发生。最近，我们发现CHIBBY 家族成员Cby1-like(Cby1L，也被称为Cby3)在 睾丸。我发现Cby1L和ciBAR1定位于鞭毛上的环，即鞭毛上的一种基于Septin的环结构，在 睾丸。Cby1L-/-和ciBAR1-/-雄性小鼠都是不育的，大多数精子显示出扭曲的尾巴。在……里面 此外，我发现Cby1L在物理上与ciBAR1和间隔环SEPT4相互作用。有趣的是，在 没有Cby1L或ciBAR1，环存在，但不能停在中段/主段(MP/PP) 结合部，并向后进入PP。这些数据表明，Cby1L和ciBAR1形成了一种复合体 帮助在MP/PP结合处正确定位环。尽管环带通常被绑定到 在迁移过程中，在Cby1L-/-睾丸中，环部分从鞭毛膜上分离， 这表明Cby1L参与了将环锚定到膜上的过程。总的来说，我的初步数据 提出一个工作模型，在该模型中，Cby1L和ciBAR1的复合体在准确的 环空在MP/PP结合处的定位。这项建议的总体目标是理解 Cby1L和ciBAR1在精子发生和精环定位中的分子作用为了实现这一目标， 我提出以下具体目标：1、确定Cby1L在鞭毛发生和环迁移中的作用。这就做 确定Cby1L在精子发生过程中的亚细胞定位，并研究其分子和 Cby1L-/-小鼠雄性不育的细胞学基础2，研究Cby1L/ciBAR1/Septin相互作用和 CIBAR1‘S在精子尾部分割中的作用我预测Cby1L与ciBAR1和间隔环蛋白形成一个复合体 以调节环与鞭毛膜的附着，这有助于适当的 精子尾部的区隔。同时，我将在ciBAR1中确定男性不育的原因-/-男性 老鼠。总体而言，我预计我提出的研究将揭开一种新的机制，以正确定位 纤毛蛋白的环，这有助于正确的精子尾巴分割。

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.