The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, Rockefeller University, New York, NY 10065, USA.
Department of Urology and Andrology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, China.
Endocrinology. 2022 Oct 11;163(11). doi: 10.1210/endocr/bqac130.
Microtubule affinity-regulating kinases (MARKs) are nonreceptor Ser/Thr protein kinases known to regulate cell polarity and microtubule dynamics in Caenorhabditis elegans, Drosophila, invertebrates, vertebrates, and mammals. An earlier study has shown that MARK4 is present at the ectoplasmic specialization and blood-testis barrier (BTB) in the seminiferous epithelium of adult rat testes. Here, we report the function of MARK4 and another isoform MARK2 in Sertoli cells at the BTB. Knockdown of MARK2, MARK4, or MARK2 and MARK4 by RNAi using the corresponding siRNA duplexes without apparent off-target effects was shown to impair tight junction (TJ)-permeability barrier at the Sertoli cell BTB. It also disrupted microtubule (MT)- and actin-based cytoskeletal organization within Sertoli cells. Although MARK2 and MARK4 were shown to share sequence homology, they likely regulated the Sertoli cell BTB and MT cytoskeleton differently. Disruption of the TJ-permeability barrier following knockdown of MARK4 was considerably more severe than loss of MARK2, though both perturbed the barrier. Similarly, loss of MARK2 affected MT organization in a different manner than the loss of MARK4. Knockdown of MARK2 caused MT bundles to be arranged around the cell periphery, whereas knockdown of MARK4 caused MTs to retract from the cell edge. These differences in effects on the TJ-permeability barrier are likely from the unique roles of MARK2 and MARK4 in regulating the MT cytoskeleton of the Sertoli cell.
微管亲和调节激酶（MARKs）是非受体丝氨酸/苏氨酸蛋白激酶，已知其在秀丽隐杆线虫、果蝇、无脊椎动物、脊椎动物和哺乳动物中调节细胞极性和微管动力学。先前的一项研究表明，MARK4 存在于成年大鼠睾丸生精上皮的质膜附属物和血睾屏障（BTB）中。在这里，我们报告了 MARK4 和另一种同工型 MARK2 在 BTB 处的支持细胞中的功能。使用相应的 siRNA 双链体进行 RNAi 敲低 MARK2、MARK4 或 MARK2 和 MARK4，没有明显的脱靶效应，结果表明紧密连接（TJ）-通透性屏障在支持细胞 BTB 处受到损害。它还破坏了支持细胞内的微管（MT）和肌动蛋白细胞骨架组织。尽管 MARK2 和 MARK4 具有序列同源性，但它们可能以不同的方式调节支持细胞 BTB 和 MT 细胞骨架。敲低 MARK4 后 TJ 通透性屏障的破坏比敲低 MARK2 严重得多，尽管两者都扰乱了屏障。同样，MARK2 的缺失对 MT 组织的影响与 MARK4 的缺失不同。MARK2 的敲低导致 MT 束排列在细胞周围，而 MARK4 的敲低导致 MT 从细胞边缘缩回。这些对 TJ 通透性屏障的影响差异可能来自 MARK2 和 MARK4 在调节支持细胞的 MT 细胞骨架方面的独特作用。
