Figure 6. Functional mechanotransduction is required for uptake of Rho-Pt.

Chemical or genetic inhibition of mechanotransduction leads to reduction in Rho-Pt uptake. Left-hand panels are representative control neuromasts for each treatment group (untreated controls for copper, cimetidine, quinine, and EGTA; wildtype (Wt) siblings for sputnik and mariner). Middle panels are representative treated or mutant neuromasts. Right-hand panels are quantified neuromast fluorescence after 1 hr of Rho-Pt treatment compared to controls. Inhibition of Ctr1 (by 0.25 µM copper) and Oct2 (by 400 µM cimetidine) have no significant effect on Rho-Pt uptake. In contrast, inhibition of mechanotransduction by quinine (100 µM) significantly reduced Rho-Pt uptake. Similarly, tip link breakage by EGTA (5 mM) also significantly reduced Rho-Pt uptake. sputnik and mariner mutants without functional mechanotransduction both demonstrate significant reduction in Rho-Pt uptake. Note that in conditions of reduced functional mechanotransduction, Rho-Pt signal appears to accumulate in the region of the cuticular plate and stereocilia (arrows), with reduced signal within the cell body. Scale bar for all figures = 10 µm. Error bars = SD; n = 20–30 neuromasts per treatment group; n = 6–10 neuromasts for wildtype sibs; *** p< 0.001, Student’s t-test.