MKL1 localization in primary megakaryocytes is regulated by TPO stimulation, Rho signaling, and actin dynamics. PreMegE cells were sorted from the BM of wild-type mice, transduced with a MIGR1-MKL1GFP fusion construct, and differentiated down the megakaryocyte lineage. (A) Time-lapse microscopy captured the rapid relocation (within 15 minutes) of MKL1 to the nucleus in cells treated with 100 ng/mL of mTPO. MKL1 transitions out of the nucleus 45 minutes after the initial nuclear accumulation. (B) MKL1 nuclear accumulation is driven by calpeptin-induced Rho activation. (C) MKL1 subcellular localization in primary cells is affected by organization of the actin cytoskeleton. MKL1 accumulates in the nucleus in response to jasplakinolide and cytochalasin D, but not in control cells exposed to DMSO alone or to latrunculin B. Time elapsed is indicated in hours and minutes (hh:mm) and a 30μM scale bar is indicated. (D) Quantification of the percentage of cells with indicated MKL1 subcellular localization in the experiments shown in panels A through C. Data were quantified from 2 independent experiments, with 7 fields per condition and at least 3 cells per field. Error bars indicate the SEM.