Figure 5. Mito-Ca²⁺ regulates ribbon formation.

(A-C) Representative images of immature neuromasts (3 dpf) immunostained with Ribeye b (magenta, ribbons) and MAGUK (green, postsynapses) after a 1 hr 0.1% DMSO (A), 2 μM Ru360 (B) or 10 µM Ru360 (C) treatment. Insets show three representative synapses (white squares) for each treatment. D-E, Scatter plot show quantification of synapse number (D), and ribbon area (E) in controls and in treatment groups. (F) Side-view of 2 hair cells (white outline) shows synaptic ribbon (three magenta asterisks in each cell) and extrasynaptic Ribeye b aggregates after a 1 hr 0.1% DMSO or 10 μM Ru360 treatment. (G) Quantification of extrasynaptic Ribeye puncta. N ≥ 12 neuromasts per treatment. Error bars in D-E and G represent SEM. An unpaired t-test was used in D and a Welch’s unequal variance t-test was used in E and G, *p<0.05, **p<0.01, ****p<0.0001. Scale bar = 5 µm in A, 2 µm in insets and F.

Figure 5—figure supplement 1. Ribbon and postsynapse size in immature ALL neuromasts.

(A-B) Scatter plots show that ribbon areas (A) and postsynaptic density areas (B) within the same fish are similar between immature anterior lateral-line (ALL) and posterior lateral-line (PLL) neuromasts. (C) Scatter plots show ribbon areas in controls and after a 1 hr treatment with 2 µM Ru360 are larger in immature hair cells within the ALL. Ribbon sizes of untreated anterior lateral-line hair cells are from the same data as A and C, n ≥ 10 neuromasts per treatment; error bars represent SEM; and a Welch’s unequal variance t-test was used for comparisons. *p<0.05.

Figure 5—figure supplement 2. MCU and Ca_V1.3 block do not impact postsynapse size.

(A) Quantification of postsynapse size assayed by MAGUK immunolabel in mature neuromasts indicate the treatments with 10 µM isradipine, 2 µM Ru360 and 10 µM Ru360 do not significantly alter postsynapse size compared to controls, n ≥ 9 neuromasts per treatment. Error bars represent SEM. A Welch’s unequal variance t-test was used for comparisons.