(A) Rosette development in wild-type S. rosetta was inhibited in the presence of 50 µg/ml anti-Rtls antibody, leading to a significant reduction in the percentage of cells in rosettes (one-tailed t test, p < 0.05) as compared to BSA, pre-immune serum, and IgG negative controls. Error bars show standard deviation. (B–C) The localization pattern of cell-associated Rtls differs between wild-type rosettes, chains, and single cells. (B) In rosettes, Rtls (cyan) was detected as a thick layer associated with the basal poles of the cells. Commonly, a gap was observed in the Rtls staining between one pair of neighboring cells in each rosette (arrow). The collar microvilli and filopodia were stained with phalloidin (red) and anti-tubulin staining (white) was used to highlight the cell body and flagellum. (C) Rtls localization in (1) wild-type rosettes, (2) wild-type chains, (3) wild-type single cells, and (4) Rosetteless mutant single cells. In single cells and chains imaged as in Figure 6B (‘Rtls’, laser intensity = 2.0, zoom = 2.5, gain = 544), Rtls signal was nearly undetectable. However, when imaged with a higher photomultiplier gain (‘Rtls–high gain’, laser intensity = 2.0, zoom = 2.5, gain = 750), Rtls was detected in membrane-associated patches (arrowheads) in wild-type single cells and chains, but not in Rosetteless cells. Wild-type single cells and chains frequently also had immunoreactive material deposited on the slide adjacent to the cells (asterisk). All cell types showed faint, diffuse fluorescence throughout the cell body, but this was likely the result of non-specific staining (Figure 6—figure supplement 2). Scale bars = 5 µm.