Primary cilia of inner ear hair cells do not respond to mechanical stimuli

Abstract

Hair cells, the sensory cells of the inner ear, carry a bundle of 30–300 specialized actin‐based microvillus‐like projections called stereocilia. Tiny filamentous ‘tip links’ connect the stereocilia and transmit the force produced by sub‐nanometer movements to Ca2+‐permeable mechanoelectrical transduction (MET) channels at the tips of stereocilia—likely encoded by TMC1 and TMC2 genes.Adjacent to the tallest stereocilia is the kinocilium—a single nonmotile, (9+2) microtubule‐based true cilium. In chicken hair cells, kinocilia are connected to stereocilia by the same proteins as the tip links, raising the question of whether kinocilia also have MET channels. Indeed, TMC1 protein has been reported in kinocilia of mouse hair cells. In developing zebrafish hair cells, moreover, Ca2+ imaging experiments suggest that, prior to the onset of the normal MET, kinocilia mediate a mechanosensitivity elicited by reverse deflections of the hair bundle. Kinocilia and kinocilial links are required for a deflection‐dependent rise in cytoplasmic [Ca2+] in zebrafish.We used fast Ca2+ imaging to explore the mechanosensitivity of kinocilia on inner and outer hair cells of the mouse cochlea. We developed a transgenic mouse, Arl13b‐mCherry‐GECO1.2, which expresses a genetically‐encoded calcium indicator (GECI) in all primary cilia and, to a lesser extent, in stereocilia. We compared [Ca2+] changes in these two structures (stereocilia and kinocilia) of similar geometry (~5 μm length and 300–600 nm diameter). During hair‐cell development (E14‐P3), we used a swept‐field confocal with millisecond frame time to examine both hair‐cell kinocilia and primary cilia of nearby supporting cells (a negative control). With fast acquisition we could distinguish Ca2+ entry directly into cilia from Ca2+ that back‐diffused from cytoplasm. The fused mCherry and GECO fluorophores allowed ratiometric Ca2+ measurement. We observed robust deflection‐induced Ca2+ influx in stereocilia, but not in kinocilia, consistent with earlier measurements.Because kinocilia lack Ca2+ influx with motion, we went on to measure responses to flow in primary cilia in tissues upon which this hypothesis has been based: in cultured kidney epithelial cells, kidney thick ascending tubules, embryonic node crown cells, mouse embryonic fibroblasts, and MLOY‐4 and OCY454 osteocyte‐like cell lines. We found a complete lack of mechanically induced calcium increases in these primary cilia. Rapid Ca2+ influx specifically into cilia was not observed with physiological—or even superphysiological—levels of fluid flow in any of these cells, suggesting that primary cilia do not respond to mechanical forces via calcium signaling. We conclude that the Ca2+‐responsive mechanosensor hypothesis of primary cilia should be abandoned.Support or Funding InformationDPC: NIH 5R01 DC000304 and HHMI; DEC: HHMI.