Activated STAT1 Transcription Factors Conduct Distinct Saltatory Movements in the Cell Nucleus

The activation of STAT transcription factors is a critical determinant of their subcellular distribution and their ability to regulate gene expression. Yet, it is not known how activation affects the behavior of individual STAT molecules in the cytoplasm and nucleus. To investigate this issue, we injected fluorescently labeled STAT1 in living HeLa cells and traced them by single-molecule microscopy. We determined that STAT1 moved stochastically in the cytoplasm and nucleus with very short residence times (<0.03 s) before activation. Upon activation, STAT1 mobility in the cytoplasm decreased similar to 2.5-fold, indicating reduced movement of STAT1/importin alpha/beta complexes to the nucleus. In the nucleus, activated STAT1 displayed a distinct saltatory mobility, with residence times of up to 5 s and intermittent diffusive motion. In this manner, activated STAT1 factors can occupy their putative chromatin target sites within similar to 2 s. These results provide a better understanding of the timescales on which cellular signaling and regulated gene transcription operate at the single-molecule level.