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Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

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Relative presence of Oc90, otolin and KSPG in neonatal B6 otoconia.(A, C) Abundant presence of Oc90 in otoconia crystals (labeled as ‘O’). (D, F) Otolin antibody showed staining in both the sensory epithelium (labeled as ‘SE’) and otoconia. (B, C, E, F) KSPG had a low level in the crystals but showed intense staining in the otoconia membrane (labeled as ‘OM’). O, otoconia; OM, otoconia membrane; SE, sensory epithelium; HC, hair cells; SC, supporting cells.
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pone-0020498-g002: Relative presence of Oc90, otolin and KSPG in neonatal B6 otoconia.(A, C) Abundant presence of Oc90 in otoconia crystals (labeled as ‘O’). (D, F) Otolin antibody showed staining in both the sensory epithelium (labeled as ‘SE’) and otoconia. (B, C, E, F) KSPG had a low level in the crystals but showed intense staining in the otoconia membrane (labeled as ‘OM’). O, otoconia; OM, otoconia membrane; SE, sensory epithelium; HC, hair cells; SC, supporting cells.

Mentions: As the main Xenopus saccular otoconin Oc22 [40] only has one sPLA2-like domain and does not have the N- or C-terminal region of Oc90, we predicted that a single sPLA2-like domain of Oc90 will be able to bind otolin and, therefore, did not test each Oc90 domain alone. Instead, we further tested whether Oc90 or otolin interacts with KSPG, another otoconia component of low to intermediate abundance. Because Western blotting of KSPG tends to produce a smear due to non-homogeneous degree of glycosylation, we used protein A agarose beads to bind an antibody for KSPG, followed by incubation with KSPG and Oc90 (or otolin) extracted from otoconia, vestibular epithelia or transfected cells. As shown in the supplemental Figure S1, the co-IP reaction produced a weak band of Oc90 (or otolin) in the reaction containing anti-KSPG+KSPG+Oc90 (or otolin), but not in the reaction containing mouse IgG+KSPG+Oc90 (or otolin) control, therefore, the reaction was likely specific albeit weak. KSPG is present in Oc90 otoconia and vestibule in a way similar to that in wt tissues [9], further suggesting weak interactions between these two components. The weak interaction between Oc90 (or otolin) and KSPG is also consistent with the observation that KSPG is low in the crystals but abundant in the otoconial membrane (OM) (Figure 2), whereas the expression of both Oc90 and otolin is higher in the crystals than the OM. Based on our previous mass spectrometry data [10], Oc90 is much more abundant in the crystals than otolin, which is evident here by the fluorescence intensity in the Figure 2. Different tissue sections were used for Oc90 and otolin immunostaining simply because of their availability at the time of the experiment; based on previous observation [10], the utricle and saccule have similar patterns of presence of the two proteins.


Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Relative presence of Oc90, otolin and KSPG in neonatal B6 otoconia.(A, C) Abundant presence of Oc90 in otoconia crystals (labeled as ‘O’). (D, F) Otolin antibody showed staining in both the sensory epithelium (labeled as ‘SE’) and otoconia. (B, C, E, F) KSPG had a low level in the crystals but showed intense staining in the otoconia membrane (labeled as ‘OM’). O, otoconia; OM, otoconia membrane; SE, sensory epithelium; HC, hair cells; SC, supporting cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105080&req=5

pone-0020498-g002: Relative presence of Oc90, otolin and KSPG in neonatal B6 otoconia.(A, C) Abundant presence of Oc90 in otoconia crystals (labeled as ‘O’). (D, F) Otolin antibody showed staining in both the sensory epithelium (labeled as ‘SE’) and otoconia. (B, C, E, F) KSPG had a low level in the crystals but showed intense staining in the otoconia membrane (labeled as ‘OM’). O, otoconia; OM, otoconia membrane; SE, sensory epithelium; HC, hair cells; SC, supporting cells.
Mentions: As the main Xenopus saccular otoconin Oc22 [40] only has one sPLA2-like domain and does not have the N- or C-terminal region of Oc90, we predicted that a single sPLA2-like domain of Oc90 will be able to bind otolin and, therefore, did not test each Oc90 domain alone. Instead, we further tested whether Oc90 or otolin interacts with KSPG, another otoconia component of low to intermediate abundance. Because Western blotting of KSPG tends to produce a smear due to non-homogeneous degree of glycosylation, we used protein A agarose beads to bind an antibody for KSPG, followed by incubation with KSPG and Oc90 (or otolin) extracted from otoconia, vestibular epithelia or transfected cells. As shown in the supplemental Figure S1, the co-IP reaction produced a weak band of Oc90 (or otolin) in the reaction containing anti-KSPG+KSPG+Oc90 (or otolin), but not in the reaction containing mouse IgG+KSPG+Oc90 (or otolin) control, therefore, the reaction was likely specific albeit weak. KSPG is present in Oc90 otoconia and vestibule in a way similar to that in wt tissues [9], further suggesting weak interactions between these two components. The weak interaction between Oc90 (or otolin) and KSPG is also consistent with the observation that KSPG is low in the crystals but abundant in the otoconial membrane (OM) (Figure 2), whereas the expression of both Oc90 and otolin is higher in the crystals than the OM. Based on our previous mass spectrometry data [10], Oc90 is much more abundant in the crystals than otolin, which is evident here by the fluorescence intensity in the Figure 2. Different tissue sections were used for Oc90 and otolin immunostaining simply because of their availability at the time of the experiment; based on previous observation [10], the utricle and saccule have similar patterns of presence of the two proteins.

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

Show MeSH
Related in: MedlinePlus