Fig. 5.

CIB2 is essential for mechanotransduction in the auditory hair cells. a, b Maximum intensity projections of Z-stacks of confocal fluorescent images (left) and corresponding DIC images (right) of control (a) and Cib2 ^tm1a/tm1a (b) cultured organ of Corti explants imaged after exposure to 3 μM of FM1-43 for 10 s. The samples were dissected at P5 and kept 2 days in vitro (P5 + 2div). Scale bar: 20 µm. c SEM images of IHCs in acutely isolated organ of Corti explants from wild-type (left), Cib2 ^F91S/F91S (middle), and Cib2 ^tm1a/tm1a (right) mice. Insets show the tip links at high magnification in the areas indicated by dashed boxes. Scale bars are 0.5 µm and 200 nm in the insets (d). Experimental setup for MET current recordings. Positive pressure in fluid-jet deflects hair bundle toward kinocilium and activates “conventional” MET channels gated by tip link tension, while negative pressure closes these channels but may activate “reverse-polarity” currents at certain conditions^{51, 52}. Scale bar is 10 µm (e, f) MET current traces (e) and average MET current (Mean ± SEM) (f) to the graded deflections of the hair bundles with fluid-jet (bottom traces in e) in wild-type (black and grey), Cib2 ^F91S/F91S (red), and Cib2 ^tm1a/tm1a (magenta) IHCs. Statistical significance is indicated with asterisks: **p < 0.01, ***p < 0.001 (Student’s t-test). g MET responses produced by a sinusoidal fluid-jet stimulus (bottom) in control Cib2 ^F91S/+ (top traces) and Cib2 ^F91S/F91S (middle traces) IHCs at different holding potential ranging from −104 to +76 mV (indicated by the traces). Age of the IHCs in e–g: P4–P7. h Reverse-polarity current in the OHCs of Cib2 ^tm1a/tm1a mice at different developmental ages indicated by the traces. Note that the current is activated by negative but not positive bundle deflections. i Average values (Mean ± SEM) of normal and reverse-polarity currents in Cib2 ^tm1a/tm1a OHCs at different ages. Number of cells is indicated by each data point.