In vitro calcite crystal morphology is modulated by otoconial proteins otolin-1 and otoconin-90

Abstract

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia.

Figure 1. Images of rmOtolin-1 molecules visualized by electron microscopy after rotary shadowing.

rmOtolin-1 molecules were adsorbed on mica and visualized by electron microscopy after rapid freezing, freeze-drying, and rotary shadowing. The interaction of the C1q domains is evident and similar to the interactions observed for collagen X by Kwan, et al. . Scale Bar, 100 nm.

Figure 2. In situ AFM height images (3.0×3.0 µm) exhibiting absorption of rmOC90 and rhOtolin-1 on freshly cleaved mica.

(A) rmOC90 sustains cluster formation randomly distributed on a mica surface after 48 hours of incubation. (B) rhOtolin-1 self assembled into a honeycomb network on a mica surface after 48 hours of incubation. rhOtolin-1 fibril height measurements reveal the presence of monomeric (arrow 1), dimeric (arrow 2) and trimeric (arrow 3) components. (C) An enlarged area of the matrix from panel B.

Figure 3. Scanning Electron Microscope Images of calcite modified by rhOtolin-1 alone or in combination with rmOC90.

Varying concentrations of rhOtolin-1 and rmOC90 protein, (A) 0 nM rhOtolin-1 and rmOC90; (B) 1000 nM rmOC90; (C) 667 nM rhOtolin-1; (D) 667 nM rhOtolin-1+500 nM rmOC90 were dissolved in 7.5 mM CaCl₂ growth solution. Crystals were grown for 48 hours by slow evaporation of NH₄HCO₃ into growth solution. Crystals were examined with JEOL JSM 6320F Field Emission scanning electron microscopy. Scale Bar, A, C, and D: 25 µm; B: 10 µm.

Figure 4. Scanning Electron Microscope Images of protein concentration dependent calcite modification by rmOC90.

Varying concentrations of rmOC90 protein, (A and D) 100 nM rmOC90; (B and E) 500 nM rmOC90; (C and F) 1000 nM rmOC90 were dissolved in 7.5 mM CaCl₂ growth solution. Crystals were grown for 48 hours by slow evaporation of NH₄HCO₃ into growth solution. Examination of the crystals was performed with JEOL JSM 6320F Field Emission scanning electron microscopy. Scale Bar, A–C: 1 mm; D–F: 25 µm.

Figure 5. Polymorph determination of calcium carbonate crystals modified by rhOtolin-1 alone or in combination with rmOC90.

Varying concentrations of rhOtolin-1 or rmOC90 protein, (A and B) 1000 nM rmOC90; (C and D) 667 nM rhOtolin-1; (E and F) 667 nM rhOtolin-1plus 500 nM rmOC90; (G) pure growth solution were dissolved in 7.5 mM CaCl₂ growth solution and crystals were grown for 48 hours by slow evaporation of NH₄HCO₃ into growth solution. Raman spectra were collected on pre-selected crystals of CaCO₃ with a HoloLab 5000 Raman microprobe spectrometer system.

Figure 6. AFM measurements of step propagation rate along both obtuse and acute directions versus rmOC90 or rhOtolin-1 concentration.

Normalized step speed rates are shown to exclude the system error between different samples and spirals. (A) Strong inhibition was observed with increasing concentrations of rmOC90. At the concentration of 40 nM the propagation speed was reduced to 30% of the original value. (B) Strong potentiation was observed with increasing concentrations of rhOtolin-1. At 40 nM and 80 nM rhOtolin-1 the propagation speed was increased 177% and 278% of the original value respectively. Error bars (10%) are estimated based on our previous experience with atomic force microscopy measurements , .