SEPT12 phosphorylation results in loss of the septin ring/sperm annulus, defective sperm motility and poor male fertility

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.

隔蛋白（septins）可通过形成异聚丝状体与环状结构参与众多细胞进程，这凸显了结构调控因子在隔蛋白组装过程中的核心重要性。已有研究证实，多种翻译后修饰（posttranslational modifications, PTMs）可调控酵母隔蛋白丝状体的动态变化。然而，目前对于翻译后修饰在调控哺乳动物隔蛋白组装中的作用仍知之甚少，而翻译后修饰对哺乳动物隔蛋白组装与功能的体内生物学意义更是尚未明确。本研究通过质谱技术鉴定出SEPT12蛋白在Ser198位点发生磷酸化，并构建了SEPT12磷酸化模拟敲入（knock-in, KI）小鼠模型，以探究该修饰的生物学意义。纯合子敲入小鼠表现出雄性生育能力低下，具体表现为精子形态异常、运动能力缺陷，以及作为隔蛋白环状结构的精子终环（annulus）缺失。对敲入小鼠睾丸组织切片的免疫组化分析显示，SEPT12磷酸化会抑制终环生物发生过程中的隔蛋白环状组装。本研究同时发现，SEPT12可经蛋白激酶A（PKA）催化发生磷酸化，而该磷酸化修饰会干扰SEPT12聚合形成复合物与丝状体。综上，本研究数据表明，SEPT12磷酸化会抑制其自身丝状体的形成，进而导致精子终环/隔蛋白环状结构缺失以及雄性生育能力低下。因此，本研究首次提供了体内遗传学证据，阐明了隔蛋白磷酸化在隔蛋白组装、细胞功能发挥及其生理学意义中的关键作用。