Fig. 9

Hair bundle localization of MET complex proteins Lhfpl5a, Pcdh15a, Tmie, Tmc1, and Tmc2b in mercury mutants.

(A–E) Representative images of (A) anti-Pcdh15a (Sibs and tomt^nl16n = 4 each), (B) GFP-Lhfpl5a (Sibs and tomt^tk256cn = 12 each), (C) Tmie-HA (Sibs and tomt^tk256cn = 15 each), (D) Tmc1-GFP (Sibs n = 17; tomt^tk256cn = 10), and (E) Tmc2b (Sibs n = 16; tomt^tk256cn = 12 in lateral cristae hair bundles at 4–5 dpf. Images in B, D, and E are from live larvae, while those in A and C are from immunolabeled, fixed specimens with phalloidin-labeled actin shown in magenta. (F) Optical sections through lateral cristae sensory patches showing the absence of Tmc1-GFP fluorescence specifically in the hair bundles of tomt^tk256c mutants, whereas GFP signal is present in the cells bodies. (G) Tomt can restore Tmc2b-GFP localization to the hair bundle of mercury mutant hair cells. Tmc2b-GFP fluorescence in neuromast hair cells of a 4 dpf Tg(myo6b:tmc2b-GFP) wild-type sibling (top) and tomt^nl16 mutant (bottom) transiently expressing tomt-p2a-nls-mCherry. Note the presence of Tmc2b-GFP only in the Tomt-P2A-nls-mCherry expressing cells of the tomt^nl16 mutant (yellow arrows, n = 3 individuals; 12 cells). (H) Representative images of Tmc2b-GFP (left panels) and merged GFP and DIC channels (right panels) in the lateral cristae of 4 dpf siblings (n = 6; top) and pcdh15a^th263b mutants (n = 6; bottom). White brackets indicated the hair bundle region of the hair cells. Scale bars = 2 µm in A-E, 5 µm in F-H.