Abstract
Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca2+-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.
Highlights
Hair cells are specialized epithelial sensory cells in the inner ear, transducing mechanical stimuli into electrical signals in the process of hearing and balance [1]
Hair cells are surrounded by supporting cells on their basolateral sides, with tight junctions sealing the borders, such that only the apical surface is accessible in a dissected explant
While the apical surface is a site of active endocytosis, only very small vesicles, about 100 nm in size, are involved; this size is too small for typical DNA complexes
Summary
Hair cells are specialized epithelial sensory cells in the inner ear, transducing mechanical stimuli into electrical signals in the process of hearing and balance [1]. Mechanoelectrical transduction occurs within the hair bundle, the unique organelle structure on the apical end of a hair cell. Many proteins critical for mechanotransduction, such as plasma membrane Ca2+-ATPase (PMCA) and harmonin, reside at discrete bundle locations that dictate their roles in mechanotransduction [2,3]. Many proteins have been identified in bundles by mass spectrometry [4], precise locations of many of them remains unknown. Immunocytochemistry works well to localize proteins; this method depends on antibodies of high specificity and affinity, which are not always available. While transient transfection of hair cells with fluorescent fusion proteins provides an alternative method that is relatively quick and independent of antibodies, most transfection methods have failed for hair cells [5]
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