The protocadherin-15-LHFPL5 tip link complex is a heterotetrameric assembly in hair cell stereocilia.

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by a MT complex located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remain unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3 to 60 amol per cochlea or utricle, well below the detection limit of most biochemical assays used to characterize macromolecular complexes. Here we describe the optimization of two single-molecule assays, single-molecule pull-down (SiMPull), and single-molecule array (SiMoA), to study the composition and quantity of the native MT subunits, protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). We demonstrate that these assays are capable of detecting and quantifying amol quantities of native protein derived from mouse cochlea and utricle. Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex.