Banerjee Trishit, Jibiki Kazuya, Sugasawa Hinata, Kanbayashi Saori, Niikura Taiki, Mano Eriko, Chaen Shigeru, Kodama Takashi S, Takahashi Satoshi, Yasuhara Noriko, Kamagata Kiyoto
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai, 980-8577, Japan; Department of Chemistry, Graduate School of Science, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai, 980-8577, Japan.
Department of Biosciences, College of Humanities and Sciences, Nihon University, Sakurajosui 3-25-40, Setagaya-ku, Tokyo, 156-8550, Japan.
Biochem Biophys Res Commun. 2025 Feb 8;748:151320. doi: 10.1016/j.bbrc.2025.151320. Epub 2025 Jan 11.
Importin α is a crucial player in the nucleocytoplasmic transport of nuclear localization signal (NLS)-containing cargo proteins and is suggested to bind to DNA directly. We hypothesized that importin α, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin αs fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single-molecule data demonstrated importin α diffuses along DNA in fast and slow mobility modes. The diffusion by importin α in the fast mobility mode did not depend on salt concentration, suggesting sliding motion with continuous contact with DNA. The sliding was supported by restricted diffusion of importin α in Cas9 obstacles bound to DNA. Next, we tested whether importin α can transport a cargo molecule along DNA. Two-color imaging data established that importin α co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin β1 together with RanGTP significantly enhanced the DNA binding of importin α and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin β1/RanGTP are involved in the switching of importin α/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin α assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin α along DNA. The relatively faster sliding by importin α allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA.
输入蛋白α是含核定位信号（NLS）的货物蛋白核质运输中的关键参与者，并且被认为可直接与DNA结合。我们推测，输入蛋白α在与DNA结合后，可能会通过滑动或跳跃沿着DNA移动。我们使用单分子荧光显微镜和单链DNA阵列研究了与AcGFP融合的输入蛋白α沿着DNA的运动动力学。单分子数据表明，输入蛋白α以快速和慢速迁移模式沿着DNA扩散。输入蛋白α在快速迁移模式下的扩散不依赖于盐浓度，这表明其为与DNA持续接触的滑动运动。输入蛋白α在与DNA结合的Cas9障碍物中的受限扩散支持了这种滑动。接下来，我们测试了输入蛋白α是否能沿着DNA运输货物分子。双色成像数据证实，输入蛋白α与作为模型货物的SV40 TAg-NLS一起沿着DNA共同滑动。我们发现，输入蛋白β1与RanGTP一起可显著增强输入蛋白α与DNA的结合以及将模型货物TRIM28募集到DNA上，这表明输入蛋白β1/RanGTP参与了输入蛋白α/货物从核运输途径向DNA滑动的转换。单分子和体内免疫荧光测定表明，输入蛋白α有助于将TRIM28积累在核染色质区域内。因此，我们展示了关于输入蛋白α沿着DNA的滑动动力学和货物运输的新发现。输入蛋白α相对较快的滑动使得货物蛋白能够有效地递送至其靶位点，即使是在长基因组DNA上。
