Evolutionary proteomics uncovers ciliary signaling components

Monika Abedin Sigg; Tabea Menchen; Jeffery Johnson; Chanjae Lee; Semil P. Choksi; Galo Garcia; Henriette Busengdal; Gerard Dougherty; Petra Pennekamp; Claudius Werner; Fabian Rentzsch; Nevan Krogan; John B. Wallingford; Heymut Omran; Jeremy F. Reiter

We discovered that dibucaine, a compound used to isolate flagella from Chlamydomonas 146 and Tetrahymena, caused choanoflagellate cells in rosette colonies to release their flagella 147 (Thompson et al., 1974; Witman, 1986) . In Chlamydomonas, diabucaine severs cilia distal to the 148 basal body and transition zone (Sanders and Salisbury, 1994) . After amputating flagella from 149 colonial choanoflagellates ( Figure 1B) , we used sucrose gradient centrifugation and fractionation 150 to separate microvilli and cell bodies from isolated cilia. Again, immunofluorescent imaging and 151 immunoblot analysis revealed that the ciliary fraction was highly enriched for TUB ac , whereas 152 the microvilli fraction was enriched for Actin ( Figure 1G and H) . 153