| First Author | Pacentine IV | Year | 2021 |
| Journal | Sci Rep | Volume | 11 |
| Issue | 1 | Pages | 23855 |
| PubMed ID | 34903829 | Mgi Jnum | J:332716 |
| Mgi Id | MGI:6836518 | Doi | 10.1038/s41598-021-03365-x |
| Citation | Pacentine IV, et al. (2021) Cy3-ATP labeling of unfixed, permeabilized mouse hair cells. Sci Rep 11(1):23855 |
| abstractText | ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced-but did not disappear altogether-in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15a(sh2/sh2) stereocilia as compared to Myo15a(sh2)/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ. |
