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The reporter mT/mG mice expressing a membrane-targeted fluorescent protein are becoming widely used to study the auditory and vestibular system due to its versatility. Here we show that high expression levels of the fluorescent mtdTomato reporter affect the function of the sensory hair cells and the auditory performance of mT/mG transgenic mice. Auditory brainstem responses and distortion product otoacoustic emissions revealed that adult mT/mG homozygous mice are profoundly deaf, whereas heterozygous mice present high frequency loss. We explore whether this line would be useful for studying and visualizing the membrane of auditory hair cells by airyscan super-resolution confocal microscopy. Membrane localization of the reporter was observed in hair cells of the cochlea, facilitating imaging of both cell bodies and stereocilia bundles without altering cellular architecture or the expression of the integral membrane motor protein prestin. Remarkably, hair cells from mT/mG homozygous mice failed to uptake the FM1-43 dye and to locate TMC1 at the stereocilia, indicating defective mechanoelectrical transduction machinery. Our work emphasizes that precautions must be considered when working with reporter mice and highlights the potential role of the cellular membrane in maintaining functional hair cells and ensuring proper hearing.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mouse, a deficiency of HGF expression limited to the auditory system, or over-expression of HGF, cause neurosensory deafness. In human, noncoding variants inHGFare associated with nonsyndromic deafnessDFNB39. However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10bp deletion (del10) inHgf, which is located in the 3’UTR of a conserved short isoform (Hgf/NK0.5). Mice homozygous for del10 exhibit moderate-to-profound hearing loss at four weeks of age as measured by pure-tone auditory brainstem responses (ABRs). The wild type +80 millivolt endocochlear potential (EP) was significantly reduced in homozygous del10 mice compared to wild type littermates. In normal cochlea, EPs are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation inHgfleads to dysfunctional ion homeostasis in the SV and a loss of EP, recapitulating human DFNB39 deafness.Significance StatementHereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously we reported that human deafness DFNB39 is associated with noncoding variants in the 3’UTR of a short isoform ofHGFencoding hepatocyte growth factor. For normal hearing, HGF levels must be fined-tuned as an excess or deficiency of HGF cause deafness in mouse. Using aHgfmutant mouse with a small 10 base pair deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

SUMMARYProtein-protein interactions (PPIs) regulate signal transduction and cellular behavior, yet studying PPIs within live cells remains fundamentally challenging. We have miniaturized the affinity pulldown, a gold-standard PPI interrogation technique, for use within live cells. Our assay hijacks endogenous myosin motors to forcibly traffic, or pulldown, macromolecular complexes within the native cytosolic environment. Macromolecules captured by nanoscale pulldown (NanoSPD) are optically interrogated in situ by tagging individual protein components. Critically, continuous motor trafficking concentrates query complexes into nanoscopic subcellular compartments, providing fluorescence enhancement and allowing nanoscale pulldowns to be visualized and quantified by standard microscopy. Nanoscale pulldown is compatible with nuclear, membrane-associated and cytoplasmic proteins and can investigate functional effects of protein truncations or amino acid substitutions. Moreover, binding hierarchies in larger complexes can be quickly examined within the natural cytosol, making nanoscale pulldown a powerful new optical platform for quantitative high-content screening of known and novel PPIs that act within macromolecular assemblies.