Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Abstract

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

Stereocilia morphogenesis and maintenance through regulation of actin stability

“…Myosin IIIa and myosin IIIb also localize to stereocilia tips along with their binding partner, espin-1, which is the longest splice isoform produced from the transcriptionally regulated Espn gene. Stereocilia are thin and unstable in hair cells lacking all espin isoforms while selective knockout of the espin-1 isoform also results in longer and thinner stereocilia, at least on extrastriolar utricular hair cells [6,17]. All espin isoforms have an actin bundling domain that is necessary to form stable stereocilia [56].…”

A link between planar polarity and staircase-like bundle architecture in hair cells

“…How the bundle adopts its staircase pattern, however, remains largely unclear. Resident bundle proteins such as Myo15a and Myo3a, and their cargo delivered to stereocilia tips, are required to various extents for graded heights and concurrent graded amounts of some proteins at tips across rows (Belyantseva et al, 2003(Belyantseva et al, , 2005Ebrahim et al, 2016;Lelli et al, 2016;Manor et al, 2011;Mburu et al, 2003;Probst et al, 1998;Salles et al, 2009). …”

Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry

“…We have used both shotgun (Francis, Krey, Krystofiak, Cui, Nanda, Xu et al, 2015; Krey et al, 2015; Wilmarth et al, 2015; Ebrahim, Avenarius, Grati, Krey, Windsor, Sousa et al, 2016; Krey, Drummond, Foster, Porsov, Vijayakumar, Choi et al, 2016) and targeted mass spectrometry (Krey et al, 2016; Ebrahim et al, 2016) to characterize mouse utricle proteins; similar methods are used with chick and rat hair bundles (Fig. 1B–G).…”

“…Details of sample loading, chromatography, electrospray ionization, and mass spectrometry are described elsewhere (Shin et al, 2013; Krey, Wilmarth, Shin, Klimek, Sherman, Jeffery et al, 2014; Francis et al, 2015; Wilmarth et al, 2015; Krey et al, 2015; Krey et al, 2016; Ebrahim et al, 2016). Data from these ThermoFisher instruments are saved as RAW files, which are then analyzed using the PAW and MaxQuant pathways described below.…”

“…Data for A–C is from ProteomeXchange PXD002167, while data for G–I is from Ebrahim et al (2016). (A) Examples of three matched LASLPAWR spectra, all from a P23 mouse hair-bundle replicate, and the summed intensities of the matched ions.…”