Dimitra Athanasiadou (1), Wenge Jiang (1,9), Natalie Reznikov (2), Alejandro B. Rodríguez-Navarro (3), Roland Kröger (4), Matthew Bilton (5), Alicia González-Segura (6), Yongfeng Hu (7), Valentin Nelea (1), Marc D. McKee (1,8)
Journal of Structural Biology, 210, Issue 2, May 2020. DOI: 10.1016/j.jsb.2020.107489

Keywords

Biomineralization, Otoconia, Osteopo, Nanostructure, Tomography

Abstract

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy – both methods being sensitive to the detection of crystalline and amorphous forms in the sample – showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

How Our Software Was Used

Dragonfly was used to create a reconstructed movie of a series of X-ray frames, which was collected over 360-degrees of rotation.

Author Affiliation

(1) Faculty of Dentistry, McGill University, Montreal, QC, Canada H3A 0C7.
(2) Object Research Systems Inc., Montreal, QC, Canada H3C 1M4.
(3) Departamento de Mineralogía y Petrología, Universidad de Granada, Granada, Spain 18002.
(4) Department of Physics, University of York, York, UK YO10 5DD.
(5) Imaging Centre at Liverpool, University of Liverpool, Liverpool, UK L69 3GL.
(6) Centro de Instrumentación Científica, Universidad de Granada, Granada, Spain 18002.
(7) Canadian Light Source, University of Saskatchewan, Saskatoon, SK, Canada S7N 2V3.
(8) Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada H3A 0C7.
(9) Department of Chemistry, Tianjin Key Laboratory of Molecular Optoelectronic Sciences, and Tianjin Collaborative Innovation Center of Chemical Science & Engineering, Tianjin University, Tianjin, P. R. China, 300072.