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Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells
Dale W. Hailey, … , Edwin W. Rubel, David W. Raible
Dale W. Hailey, … , Edwin W. Rubel, David W. Raible
Published December 19, 2016
Citation Information: J Clin Invest. 2017;127(2):472-486. https://doi.org/10.1172/JCI85052.
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Research Article Cell biology

Fluorescent aminoglycosides reveal intracellular trafficking routes in mechanosensory hair cells

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Abstract

Aminoglycosides (AGs) are broad-spectrum antibiotics that are associated with kidney damage, balance disorders, and permanent hearing loss. This damage occurs primarily by killing of proximal tubule kidney cells and mechanosensory hair cells, though the mechanisms underlying cell death are not clear. Imaging molecules of interest in living cells can elucidate how molecules enter cells, traverse intracellular compartments, and interact with sites of activity. Here, we have imaged fluorescently labeled AGs in live zebrafish mechanosensory hair cells. We determined that AGs enter hair cells via both nonendocytic and endocytic pathways. Both routes deliver AGs from the extracellular space to lysosomes, and structural differences between AGs alter the efficiency of this delivery. AGs with slower delivery to lysosomes were immediately toxic to hair cells, and impeding lysosome delivery increased AG-induced death. Therefore, pro-death cascades induced at early time points of AG exposure do not appear to derive from the lysosome. Our findings help clarify how AGs induce hair cell death and reveal properties that predict toxicity. Establishing signatures for AG toxicity may enable more efficient evaluation of AG treatment paradigms and structural modifications to reduce hair cell damage. Further, this work demonstrates how following fluorescently labeled drugs at high resolution in living cells can reveal important details about how drugs of interest behave.

Authors

Dale W. Hailey, Robert Esterberg, Tor H. Linbo, Edwin W. Rubel, David W. Raible

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Figure 3

Small molecules and mutations protect HCs from AG exposure by affecting MET and AG loading.

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Small molecules and mutations protect HCs from AG exposure by affecting ...
(A) Example images from a Z-series showing technique to quantify AG loading. myo6-GFP zebrafish have labeled LL HCs (left panels). Otsu segmentation of the GFP signal generates an HC-specific mask (right panels). This is used to quantify the Neo-TR signal in HCs (middle left panels). Scale bar: 5 μm. (B) Diverse types of small molecules protect HCs from AG exposure by inhibiting AG entry, as is seen in the sputniktj264a and marinerty220 MET mutants. Neo-TR uptake is quantified as described in A. Each graphed symbol represents 1 fish, 5 neuromasts per fish. (C) Example images from Neo-TR pulse-labeled HCs either untreated, treated with the MET inhibitor amiloride, or treated with ractopamine. Scale bar: 5 μm. (D) The MET inhibitor amiloride impedes HC entry of the MET activity indicator FM1-43 in a dose-dependent manner. Zebrafish (5 dpf) were pretreated with amiloride at the indicated concentrations for 5 minutes, exposed to amiloride plus 500 nM FM1-43 for 5 minutes, washed 3 times, and imaged. FM1-43 fluorescence in neuromasts was quantified and expressed as signal relative to background outside HCs. (E) The dose-dependent reduction of Neo-TR loading mimics the reduction in MET activity in D. (Quantified as in D, but with 50 nM Neo-TR replacing FM1-43.) Error bars for all plots: ± 1 SD.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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