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

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

The basal and lateral localization of SrSeptin2 and SrSeptin6 in rosettes is reminiscent of 233 septin localization in polarized epithelial cells (Fares et al., 1995; Spiliotis et al., 2008) , in which 234 septins interact with the positive ends of microtubules that are growing toward the basal pole 235 (Bowen et al., 2011) . In choanoflagellates, microtubules radiate down from the apical 236 microtubule organizing centers (Karpov and Leadbeater, 1998) , with the plus ends meeting at 237 the basal pole of each cell, similar to the orientation of microtubule plus ends toward the basal 238 pole in animal epithelia (Meads and Schroer, 1995) . To examine if septins also interact with the 239 plus ends of microtubules in S. rosetta, we co-transfected cells with mTFP1-SrSeptin2 and the 240 tubulin marker α-tubulin-mCherry (Fig. 4G ). Fluorescence microscopy showed that septin 241 filaments intercalate between cortical microtubules at the basal pole of the cell ( laboratories. This method overcomes numerous barriers that prevented efficient DNA delivery in 252 our prior attempts using diverse methods, including standard electroporation, lipofection, 253 bombardment, and cell-penetrating peptides. A key breakthrough for this study was the 254 discovery that the extracellular coat surrounding S. rosetta might present a barrier for 255 transfection, which motivated the development of a method to gently remove the extracellular 256 material surrounding S. rosetta, thereby sensitizing cells for transfection. Additional 257 improvements to the transfection procedure, such as a step for promoting the closure of the 258 plasma membrane after electrical pulsation, were designed to address the unique challenges 259 that arise from culturing S. rosetta in sea water. Just as our method was informed by 260 approaches developed in model microeukaryotes (Chlamydomonas and yeast), the methods we 261 have established in S. rosetta will likely extend to aid gene delivery in diverse non-model marine 262