Choanoflagellate transfection illuminates their cell biology and the ancestry of animal septins

David S. Booth; Heather Szmidt-Middleton; Nicole King

Fluorescence persisted through multiple cell divisions, yet the diminishing signal in daughter 135 cells indicated that transfection was transient (Fig. S6A) . Importantly, using flow cytometry one 136 to two days after transfection, we found that ~1% of the population was reproducibly 137 transfected, and fluorescence-activated cell sorting enriched this transfected cell population 138 (Fig. S6B ). This transfection frequency is comparable to high frequency episomal transformation 139 of the model yeast Saccharomyces cerevisiae that ranges from 1 -10% (Schiestl and Electron micrographs have revealed two distinct regions in the nucleus: the darkly-stained 154 nucleolus positioned in the center and the surrounding, more lightly-stained nucleoplasm 155 (Leadbeater, 2015; Burkhardt et al., 2014) . As predicted, mCherry fused to either the carboxy 156 terminus of H3 or the amino terminus of the simian virus 40 nuclear localization signal localized 157 primarily to the S. rosetta nucleoplasm and was excluded from the cytoplasm (Fig. 3B , C) 158 (Kanda et al., 1998; Kalderon et al., 1984) . In contrast, the cytoplasmic marker EFL-mCherry 159 (Huh et al., 2003) localized to the cytosol and was excluded from the nucleus (Fig. 3D) . 160