Single-molecule microscopy reveals that importin α slides along DNA while transporting cargo molecules

Importin alpha 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 alpha, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin alpha s fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single- molecule data demonstrated importin alpha diffuses along DNA in fast and slow mobility modes. The diffusion by importin alpha 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 alpha in Cas9 obstacles bound to DNA. Next, we tested whether importin alpha can transport a cargo molecule along DNA. Two-color imaging data established that importin alpha co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin (31 together with RanGTP significantly enhanced the DNA binding of importin alpha and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin (3 1/RanGTP are involved in the switching of importin alpha/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin alpha assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin alpha along DNA. The relatively faster sliding by importin alpha allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA.