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Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula
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Hearing Research 189 (2004) 76^82 www.elsevier.com/locate/heares
Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula Marina Piscopo a , Bice Avallone a , Loredana D’Angelo a , Umberto Fascio b , Giuseppe Balsamo a , Francesco Marmo a; a
Department of Genetics, General and Molecular Biology, University of Naples ‘Federico II’, Naples, Italy b CIMA, University of Milan, Milan, Italy Received 31 July 2003; received in revised form 28 October 2003; accepted 28 October 2003
Abstract The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2þ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals. 4 2003 Elsevier B.V. All rights reserved. Key words: Calbindin D-28K; Otoconia; Inner ear; Aragonite; Podarcis sicula
1. Introduction The ability to sense orientation relative to gravity requires crystalline structures, called otoconia, composed of organic matrix and inorganic material (Henle, 1873), which are localized in the vestibular macular organs. The inorganic phase is characterized by an evolutionary trend toward deposition of crystal polymorphs of CaCO3 of increasing stability. The least stable polymorph (vaterite) is present in the primitive otoconia of the hag¢sh, aragonite predominates in amphibia and reptiles, and birds and mammals are characterized by calcite, the most stable polymorph (Carlstro«m, 1963; Marmo et al., 1983a,b; Ross et al., 1984). Calcite, aragonite and vaterite have the same chemical
* Corresponding author. Tel.: +39 (81) 2535012 (o⁄ce), +39 (81) 2535006-011 (lab.); Fax: +39 (81) 2535000. E-mail address: [email protected] (F. Marmo). Abbreviations: SDS^PAGE, sodium dodecyl sulfate^ polyacrylamide gel electrophoresis; PBS, phosphate-bu¡ered saline; CBP, calcium-binding protein; CLSM, confocal laser scanning microscope; ICT, transmitted light interference contrast
composition but di¡er from each other with respect to physical properties such as crystalline form, speci¢c gravity, refractive index and hardness (Marmo et al., 1983a,b). Crystals showing a cylindrical body with a triplanar smooth facet at each end can be identi¢ed as calcite otoconia. Those with a pseudohexagonal prismatic shape, with £at sides and end faces or a fusiform shape with rounded body and pointed end are aragonite otoconia. Vaterite crystals have the form of a biconvex lens, which is di¡erent from that found in other biological (Lowenstam and Abbott, 1975) or inorganic structures (Meyer, 1965). The organic phase of otoconia consists of proteins and carbohydrate (Wislocki and Ladman, 1955; Be'langer, 1960; Marmo et al., 1964; Ross et al., 1984; Ross et al., 1985; Gil-Loyzaga et al., 1985; Fermin et al., 1998). According to Lowenstam and Weiner (1989) the organic matrix is involved in the seeding, growth and the type of crystal polymorph formed. The biological mechanisms responsible for development, biosynthesis and maintenance of otoconia are not completely understood. The prevailing hypothesis is that otoconia are formed in the extracellular space from the calcium and carbonate ions of
0378-5955 / 03 / $ ^ see front matter 4 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00366-6
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endolymph (Marmo, 1971; Ross et al., 1985; Ross and Pote, 1984; Erway et al., 1986). Because of the low concentration of these ions in endolymph, it is believed that matrix proteins are required for the nucleation and growth of the crystals (Lim, 1980; Ross et al., 1985; Ross and Pote, 1984; Fermin et al., 1987). De Vincentis and Marmo (1966) have demonstrated a precocious incorporation of 45 Ca in the organic matrix of the otoconia by autoradiographic studies on the calcium turnover in the membranous labyrinth of developing chick embryos during the otoconia morphogenesis. It has been shown that otoconial complexes of adult mammals sequester calcium (Be'langer, 1960; De Vincentis and Marmo, 1966; Preston et al., 1975; Ross, 1979; Ross et al., 1980) as do those of fetal mice (Veenho¡, 1969) and chicken and Ca2þ -binding proteins (CBPs) have received special attention, since they modulate intracellular Ca2þ concentration. Calbindin belongs to the CBP group that acts as Ca2þ bu¡er, responsible for maintaining low levels of intracellular free Ca2þ . Calbindin D-28K has been observed at the periphery of otoconia of chick embryos and chicken, by immunohistochemistry (Balsamo et al., 2000). We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard Podarcis sicula otoconia (Piscopo et al., 2003). The otoconia of lizard P. sicula consist of calcite (Fig. 1A) and aragonite (Fig. 1B). While the saccule contains calcite crystals adjacent to the macular epithelium, in addition to a preponderance of aragonite crystals, the utricle and lagena contain only calcite crystals (Marmo et al., 1981). In order to determine the localization of calbindin D-28K, immunoblot assays with monoclonal anti-calbindin D-28K antibodies were performed on proteins extracted from utricle plus lagena otoconia in comparison with those extracted from saccule otoconia. Moreover, calcite and aragonite otoconia were analyzed by confocal laser
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scanning microscope (CLSM) analysis using the same antibodies.
2. Materials and methods 2.1. Protein extraction and analysis Twenty adult specimens of lizard P. sicula on average of 7.00 g weight each were collected in localities around Naples, Italy. The animals were maintained in the laboratory under natural conditions of light (10^13 h) and temperature (ranging from 14 to 24‡C) and then decapitated under general anesthesia with ether vapors according to the institutional animal care and use committee. Temporal bones were removed and immersed in 0.9% NaCl. Otoconia were taken with a siliconized pulled Pasteur pipette under a stereomicroscope. The upper portion of mass of otoconia was taken with great care leaving a layer on the epithelium in order to be sure of taking only otoconia and not other cells and/or hairs contaminating the preparation. Otoconia from utricle and lagena were pooled while those from saccule were collected separately. Proteins from otoconia were extracted according to Piscopo et al. (2003). Protein extraction from papilla basilaris of two adult specimens of Gallus domesticus and from saccule, utricle and lagena (previously deprived of otoconia) of two adult specimens of P. sicula was performed according to Piscopo et al. (2003). Samples were analyzed using sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^ PAGE) to determine their protein content. 12.5% polyacrylamide gels were used with a 5% stacking gel. Laemmli’s Tris^glycine bu¡er was used for electrophoresis (Laemmli, 1970). Gels were run until the tracking dye had just left the gel and stained with silver nitrate (Merril et al., 1984).
Fig. 1. Scanning electron micrographs. A: Calcite otoconia (1400U). B: Aragonite otoconia (1500U).
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Fig. 2. A: SDS^PAGE analysis of: molecular weight standards (lane M); proteins extracted from P. sicula saccule otoconia (lane Ot.S); proteins extracted from P. sicula utricle plus lagena otoconia (lane Ot.U+L); proteins extracted from P. sicula saccule, utricle, lagena, ampulla, basilaris papilla epithelia (lanes S, U, L, A, P, respectively). B: Immunoblot assay on the same samples shown in A with monoclonal anti-calbindin D-28K; protein extracted from chicken basilaris papilla (+).
2.2. Western immunoblot Immediately after electrophoresis the gel was placed in transfer bu¡er (20% methanol, 0.025 mM Tris-base, 0.129 M glycine pH 8.5) and the nitrocellulose paper (Amersham 0.45 Wm pore) was placed in a separate dish containing fresh transfer bu¡er and four pieces of ¢lter paper. Filter was removed from the transfer bu¡er and placed on top of the gel. A sandwich of the nitrocellulose and gel was placed between the presoaked ¢lter
paper and placed in a Bio-Rad Mini transblot. The transfer took place for 2 h at 100 V constant at 4‡C. The electroblot was removed from the transfer apparatus and treated as follows: (1) 3% Nidina (not fat dry milk) in 0.1 M phosphate-bu¡ered saline (PBS) pH 7.2 (1% NaCl, 0.025% KCl, 0.144% Na2 HPO4 , 0.025% K2 HPO4 ) for 60 min at 37‡C, (2) primary antibody: monoclonal anti-calbindin D-28K (mouse IgG1 isotype) (Sigma, St. Louis, MO, USA) used at a dilution of 1/2500 in the same bu¡er for 2 h at 37‡C, (3) three
C Fig. 3. CLSM micrographs of P. sicula aragonite and calcite otoconia treated with anti-calbindin D-28K and rabbit FITC-conjugated antimouse. Aragonite otoconia decalci¢ed with: A2 : dH2 O at 37‡C for 24 h (1600U); B2 : dH2 O at 37‡C for 48 h (1600U); C, E: HCl 1 N at room temperature for 2 min (1100U); H2 : HCl 1 N at room temperature for 3 min (1300U). D, F, I: Three-dimensional reconstruction of otoconia shown in C, E, H2 , respectively. G2 : Calcite otoconia decalci¢ed with dH2 O at 37‡C for 24 h (600U); A1 , B1 , G1 , H1 : ICT images of crystals shown in A2 , B2 , G2 , H2 , respectively.
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washes with 0.1 M PBS and then secondary antibody : horseradish peroxidase rabbit anti-mouse IgG (Sigma, St. Louis, MO, USA) used at a dilution of 1/1000 for 1 h at 37‡C, (4) three washes with 0.1 M PBS, (5) addition of 10 ml of methanol containing 3 mg/ml 4-chloro-1naphthol, and then addition of 10 Wl of H2 O2 30 v/v. The ¢lter was kept on constant motion until the bands appeared and then washed with double-distilled water. 2.3. CSLM Otoconia were subjected to a mild (dH2 O at 37‡C for 12^72 h) or more heavy decalci¢cation (HCl 1 N at room temperature for 2^3 min) and then treated with monoclonal anti-calbindin D-28K (mouse IgG1 isotype) (Sigma, St. Louis, MO, USA) as primary antibody and rabbit £uorescein isothiocyanate (FITC)-conjugated anti-mouse as secondary antibody, each used at a dilution of 1:1000. Fluorescence observations were carried out by a confocal microscope (Leica TCSNT) with laser argon-krypton 7^5 mW multiline. Focal series of horizontal planes of sections were monitored for FITC using the 488 nm laser line and a bandpass 590/30-nm ¢lter. Preimmune sera, instead of speci¢c antisera, were used in the negative control. 2.4. Use of animals Samples captured with authorization of 1/06/2000 N. SCN/2D/2000/9213 del Ministero dell’Ambiente.
et al., 1988; Balsamo et al., 2000). Immunoreactivity was observed only in the sample containing the proteins extracted from otoconia of saccule (Fig. 2B, lane Ot.S) and in that containing the proteins extracted from saccule epithelium (Fig. 2B, lane S). These results have been con¢rmed by CLSM analyses that have also indicated the presence of calbindin D-28K not only on the surface but also in the core of aragonite crystals. In fact in these crystals after weak decalci¢cation (24^48 h in dH2 O at 37‡C), £uorescence on the surface of crystal is evident (Fig. 3A2 , B2 ) while in those decalci¢ed with HCl 1 N for 2^3 min at room temperature, £uorescence was found in the central portion of crystal (Fig. 3C, E, H2 ). Fig. 3A1 , B1 , G1 , H1 are the corresponding images, at transmitted light interference contrast (ICT) of crystals shown in Fig. 3A2 , B2 , G2 , H2 . The crystals in Fig. 3A1 and B1 show weak erosion while Fig. 3H1 shows only the protein core of crystal following to the stronger decalci¢cation. In the calcite crystals, either weakly or strongly decalci¢ed, £uorescence was not found (Fig. 3G2 ). A three-dimensional reconstruction obtained from the superimposition of 20 optical sections shows a typical aspect of otoconia after decalci¢cation (Fig. 3D, F, I). Fig. 3D, F show the aragonite crystals after decalci¢cation with HCl 1 N for 2 min. In this case the crystals are not yet completely decalci¢ed. Calbindin D-28K is evident in the protein core of aragonite crystal after complete decalci¢cation with HCl 1 N for 3 min (Fig. 3H2 ). All negative controls did not show £uorescence.
3. Results 4. Discussion In Fig. 2A is shown the SDS^PAGE of the proteins contained in the otoconia of saccule (lane Ot.S); in the otoconia of utricle plus lagena (lane Ot.U+L); and of the proteins extracted from P. sicula saccule, utricle, lagena, ampulla and papilla epithelia (lanes S, U, L, A and P, respectively), after otoconia were removed from the saccule, utricle and lagena. The samples show similar electrophoretic patterns and all seem to present a band of about 28 kDa. Since we have already shown that the organic matrix of P. sicula otoconia contains a protein of this molecular weight corresponding to calbindin D-28K, the same samples shown in Fig. 2A were rerun and the proteins transferred on a nitrocellulose ¢lter. The immunoblotting was performed with monoclonal anti-calbindin D-28K (Fig. 2B) in order to establish if calbindin could be present in all samples or it could be typical of a particular type of crystal and/or epithelia. The proteins extracted from chicken basilaris papilla were used as positive control (+), calbindin D-28K being one of its components (Oberholtzer
Calbindin D-28K belongs to the family of the CBPs. Special attention has been paid to these proteins because they modulate cellular Ca2þ level. Calbindin is a 28 kDa vitamin D-induced protein located in the peripheral vestibular system of various vertebrates (Dechesne et al., 1988a) where it could play a role in the electrophysiological functioning of the sensory cells. Expression of calbindin D-28K was seen in the hair cells and the vestibular ganglion on gestional day (GD) 19, in nerve ¢bers on GD23 and in otoconia on GD26 of the musk shrew (Karita et al., 1999). Sans et al. (1986) have suggested that the presence of calbindin D-28K may be associated with at least three important roles of Ca2þ in vestibular hair cells: transduction phenomena, contraction of sensory apex proteins and transmitter release. Numerous calbindin D-28K immunoreactive particles were found within the otolithic membrane on the sensory hair cells layer in the squirrel monkey (Usami et
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al., 1995). These ¢ndings may indicate that the Ca2þ transport mechanism is necessary in forming and maintaining otoconia. Recently we have demonstrated the presence of calbindin D-28K in the organic matrix of P. sicula otoconia (Piscopo et al., 2003) in line with Balsamo et al. (2000) who have found calbindin D28K immunoreactivity in the periphery and also in the central portion of some otoconia of chick embryos and adult animals indicating that otoconia are dynamic structures, which undergo turnover. Now we have evidenced by immunoblot assay with monoclonal anti-calbindin D-28K the presence of calbindin D-28K only in the aragonite otoconia and this result was con¢rmed also by CLSM analyses and indirectly also by the exclusive localization of this protein in the saccule epithelium (probable place of origin of the crystals). This ¢nding can be correlated to the more speci¢c function of calcareous reserve that aragonitic otoconia carry out in many classes of vertebrates, reptiles and amphibia (Guardabassi, 1952, 1953; Marmo et al., 1981, 1983a,b). This function of aragonite otoconia is likely due to the fact that Ca2þ of aragonite crystals is more easily mobilizable as demonstrated by several studies and also by scanning electron microscopy analyses that indicate evident signs of erosion of aragonite crystals, not present in calcite otoconia, in amphibia and reptiles (Marmo et al., 1986). It is possible to hypothesize that calbindin D-28K in those crystals could act as solubilizator of Ca2þ from the reserve deposits. In fact, calbindin D-28K is one of the Ca2þ bu¡ering proteins responsible for maintaining low levels of intracellular free Ca2þ (Slepecky and Ulfendhal, 1993). Usami et al., 1995, in fact, have suggested that Ca2þ in the endolymph may be taken up into organic substances in the otoconia via calbindin. Such a mechanism may start functioning during development. Thus, it is possible to hypothesize that calbindin D-28K can be involved both in the process of mineralization of otoconia, as demonstrated by the presence of this protein in the otoconia of chick embryo (Balsamo et al., 2000) and in their decalci¢cation in the case otoconia is functioning as calcareous reserve.
References Balsamo, G., Avallone, B., Del Genio, F., Marmo, F., 2000. Calci¢cation processes in the chick otoconia and calcium binding proteins: patterns of tetracycline incorporation and calbindin D-28K distribution. Hear. Res. 148, 1^8. Be'langer, L.F., 1960. Development, structure and composition of the otolithic organs of the rat. In: R.F. Sognnaes (Ed.), Calci¢cation in Biological Systems. American Association for the Advancement of Science, Washington, DC, pp. 151^162. Carlstro«m, D., 1963. A crystallographic study of vertebrate otolithis. Biol. Bull. 125, 421^463.
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Dechesne, C.J., Thomasset, M., Brehier, A., Sans, A., 1988a. Calbindin (CaBP 28 kDa) localization in the peripheral vestibular system of various vertebrates. Hear. Res. 33, 273^278. De Vincentis, M., Marmo, F., 1966. The 45 Ca turnover in the membranous labyrinth of chick embryo during development. J. Embryol. Exp. Morphol. 15, 349^354. Erway, L.C., Purichia, N., Netzler, R., D’Amore, M., Esses, I., Levine, M., 1986. Genes, manganese, and zinc in formation of otoconia: labeling, recovery, and maternal e¡ects. Scanning Electron Microsc. 4, 1681^1694. Fermin, C.D., Igarashi, M., Yoshihara, T., 1987. Ultrastructural changes of statoconia after segmentation of the otolithic membrane. Hear. Res. 28, 23^34. Fermin, C.D., Lychakov, D., Campos, A., Hara, H., Sondag, E., Jones, T., Jones, S., Taylor, M., Meza-Ruiz, G., Martin, D.S., 1998. Otoconia biogenesis, phylogeny, composition and functional attributes. Histol. Histopathol. 13, 1103^1154. Gil-Loyzaga, P., Raymond, J., Gabrion, J., 1985. Carbohydrates detected by lectins in the vestibular organ. Hear. Res. 18, 269^272. Guardabassi, A., 1952. L’organo endolinfatico degli an¢bi anuri. Arch. Ital. Anat. Embryol. 57, 241^294. Guardabassi, A., 1953. Les sels de Ca du sac endolymphatique et les processus de calci¢cation des os pendant la metamorphose normal et expe¤rimentale chez le te¤tards de Bufo vulgaris, Rana dalmatina et Rana esculenta. Arch. Anat. Microsc. Morphol. Exp. 42, 143^167. Henle, J., 1873. Quoted by Rudinger in: Stricker, S. (Ed.), Manual of Human and Comparative Histology Vol. 3. New Sydenham Society, London, p. 122. Karita, K., Nishizaki, K., Nomiya, S., Masuda, Y., 1999. Calbindin and calmodulin localization in the developing vestibular organ of the musk shrew (Suncus murinus). Acta Otolaryngol. Suppl. 540, 16^21. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680^685. Lim, D.J., 1980. Morphogenesis and malformation of otoconia: a review. Birth Defects Orig. Artic. Ser. 16, 111^146. Lowenstam, H.A., Abbott, D.P., 1975. Vaterite: a mineralization product of the hard tissues of a marine organism (Ascidiacea). Science 188, 363^365. Lowenstam, H.A., Weiner, S., 1989. On Biomineralization. Oxford University Press, New York. Marmo, F., De Vincentiis, M., Materazzi, G., 1964. Caratterizzazione istochimica dei Mucopolisaccaridi di alcune strutture del labirinto membranoso dell’embrione di pollo. Riv. Istochim. 10, 733^742. Marmo, F., 1971. Uptake of metabolic CO2 by the otoliths of the chick embryo. Experientia 27, 1326^1327. Marmo, F., Franco, E., Balsamo, G., 1981. Scanning electron microscopic and X-ray di¡raction studies of otoconia in the Lizard Podarcis s. sicula. Cell Tissue Res. 218, 265^270. Marmo, F., Balsamo, G., Crispino, P., 1983a. Preliminary observation on the organic material of otoconial membranes in the tadpole of Rana esculenta. Acta Embryol. Morphol. Exp. 4, 202^203. Marmo, F., Balsamo, G., Franco, E., 1983b. Calcite in the statoconia of amphibians: a detailed analysis in the frog Rana esculenta. Cell Tissue Res. 233, 35^43. Marmo, F., Balsamo, G., Crispino, P., 1986. Ultrastructural aspects of the endolymphatic organ in the frog Rana esculenta. Acta Zool. 67, 53^61. Merril, C.R., Goldman, D., Van Keuren, M.L., 1984. Gel protein stains: Silver stains. In: Methods in Enzymology Vol. 104, pp. 441^447. Meyer, H.G., 1965. Bildung und Morphologie des Vaterits. Z. Kristallogr. Kristallgeom. 66, 220^242. Oberholtzer, J.C., Buettger, C., Summers, M.C., Matschinsky, F.M.,
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1988. The 28-kDa calbindin-D is a major calcium-binding protein in the basilar papilla of the chick. Proc. Natl. Acad. Sci. USA 85, 3387^3390. Piscopo, M., Balsamo, G., Mutone, R., Avallone, B., Marmo, F., 2003. Calbindin D-28K is a component of the organic matrix of lizard Podarcis sicula otoconia. Hear. Res. 178, 89^94. Preston, R.E., Johnson, L.G., Hill, J.H., Schacht, J., 1975. Incorporation of radioactive calcium into otolithic membranes and middle ear ossicles of the gerbil. Acta Otolaryngol. (Stockh.) 80, 269^275. Ross, M.D., 1979. Calcium ion uptake and exchange in otoconia. Adv. Otorhinolaryngol. 25, 26^33. Ross, M.D., Pote, K.D., 1984. Some properties of octoconia. Philos. Trans. R. Soc. Lond., B. Biol. Sci. Ross, M.D., Donovan, K., Chee, O., 1985. Otoconial morphology in space-£own rats. Physiologist 28 (6 Suppl.), 219^220. Ross, M.D., Pote, K.G., Cloke, P.L., Corson, C., 1980. In vitro 45 Ca2þ uptake and exchange by otoconial complexes in high and low Kþ /Naþ £uids. Physiologist 23 (Suppl. 6), 129^130. Ross, M.D., Pote, K.G., Perini, F., 1984. Analytical studies of the
organic material of otoconial complexes, including its amino acid and carbohydrate composition. In: Auditory Biochemistry, pp. 351^365. Sans, A., Etchecopar, B., Bhehier, A., Thomasset, M., 1986. Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28K) in the vestibular hair cells of the cat. Brain Res. 364, 190^194. Slepecky, M.B., Ulfendhal, M., 1993. Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti. Hear. Res. 70, 73^84. Usami, S., Shinkawa, H., Inoue, Y., Kanzaki, J., Anniko, M., 1995. Calbindin D-28K localization in the primate inner ear. ORL 57, 94^99. Veenho¡, V.B., 1969. The Development of Statoconia in Mice. Akademie van Wetenschappen, Afdeling Natuurkunde 2/58, No. 4. N.V. North-Holland, Amsterdam. Wislocki, G.B., Ladman, A.G., 1955. Selective and histochemical staining of the otolithic membranes cupulae and tectorial membrane of the inner ear. J. Anat. Lond. 89, 3^12.
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Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula Marina Piscopo a , Bice Avallone a , Loredana D’Angelo a , Umberto Fascio b , Giuseppe Balsamo a , Francesco Marmo a; a
Department of Genetics, General and Molecular Biology, University of Naples ‘Federico II’, Naples, Italy b CIMA, University of Milan, Milan, Italy Received 31 July 2003; received in revised form 28 October 2003; accepted 28 October 2003
Abstract The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2þ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals. 4 2003 Elsevier B.V. All rights reserved. Key words: Calbindin D-28K; Otoconia; Inner ear; Aragonite; Podarcis sicula
1. Introduction The ability to sense orientation relative to gravity requires crystalline structures, called otoconia, composed of organic matrix and inorganic material (Henle, 1873), which are localized in the vestibular macular organs. The inorganic phase is characterized by an evolutionary trend toward deposition of crystal polymorphs of CaCO3 of increasing stability. The least stable polymorph (vaterite) is present in the primitive otoconia of the hag¢sh, aragonite predominates in amphibia and reptiles, and birds and mammals are characterized by calcite, the most stable polymorph (Carlstro«m, 1963; Marmo et al., 1983a,b; Ross et al., 1984). Calcite, aragonite and vaterite have the same chemical
* Corresponding author. Tel.: +39 (81) 2535012 (o⁄ce), +39 (81) 2535006-011 (lab.); Fax: +39 (81) 2535000. E-mail address: [email protected] (F. Marmo). Abbreviations: SDS^PAGE, sodium dodecyl sulfate^ polyacrylamide gel electrophoresis; PBS, phosphate-bu¡ered saline; CBP, calcium-binding protein; CLSM, confocal laser scanning microscope; ICT, transmitted light interference contrast
composition but di¡er from each other with respect to physical properties such as crystalline form, speci¢c gravity, refractive index and hardness (Marmo et al., 1983a,b). Crystals showing a cylindrical body with a triplanar smooth facet at each end can be identi¢ed as calcite otoconia. Those with a pseudohexagonal prismatic shape, with £at sides and end faces or a fusiform shape with rounded body and pointed end are aragonite otoconia. Vaterite crystals have the form of a biconvex lens, which is di¡erent from that found in other biological (Lowenstam and Abbott, 1975) or inorganic structures (Meyer, 1965). The organic phase of otoconia consists of proteins and carbohydrate (Wislocki and Ladman, 1955; Be'langer, 1960; Marmo et al., 1964; Ross et al., 1984; Ross et al., 1985; Gil-Loyzaga et al., 1985; Fermin et al., 1998). According to Lowenstam and Weiner (1989) the organic matrix is involved in the seeding, growth and the type of crystal polymorph formed. The biological mechanisms responsible for development, biosynthesis and maintenance of otoconia are not completely understood. The prevailing hypothesis is that otoconia are formed in the extracellular space from the calcium and carbonate ions of
0378-5955 / 03 / $ ^ see front matter 4 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00366-6
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endolymph (Marmo, 1971; Ross et al., 1985; Ross and Pote, 1984; Erway et al., 1986). Because of the low concentration of these ions in endolymph, it is believed that matrix proteins are required for the nucleation and growth of the crystals (Lim, 1980; Ross et al., 1985; Ross and Pote, 1984; Fermin et al., 1987). De Vincentis and Marmo (1966) have demonstrated a precocious incorporation of 45 Ca in the organic matrix of the otoconia by autoradiographic studies on the calcium turnover in the membranous labyrinth of developing chick embryos during the otoconia morphogenesis. It has been shown that otoconial complexes of adult mammals sequester calcium (Be'langer, 1960; De Vincentis and Marmo, 1966; Preston et al., 1975; Ross, 1979; Ross et al., 1980) as do those of fetal mice (Veenho¡, 1969) and chicken and Ca2þ -binding proteins (CBPs) have received special attention, since they modulate intracellular Ca2þ concentration. Calbindin belongs to the CBP group that acts as Ca2þ bu¡er, responsible for maintaining low levels of intracellular free Ca2þ . Calbindin D-28K has been observed at the periphery of otoconia of chick embryos and chicken, by immunohistochemistry (Balsamo et al., 2000). We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard Podarcis sicula otoconia (Piscopo et al., 2003). The otoconia of lizard P. sicula consist of calcite (Fig. 1A) and aragonite (Fig. 1B). While the saccule contains calcite crystals adjacent to the macular epithelium, in addition to a preponderance of aragonite crystals, the utricle and lagena contain only calcite crystals (Marmo et al., 1981). In order to determine the localization of calbindin D-28K, immunoblot assays with monoclonal anti-calbindin D-28K antibodies were performed on proteins extracted from utricle plus lagena otoconia in comparison with those extracted from saccule otoconia. Moreover, calcite and aragonite otoconia were analyzed by confocal laser
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scanning microscope (CLSM) analysis using the same antibodies.
2. Materials and methods 2.1. Protein extraction and analysis Twenty adult specimens of lizard P. sicula on average of 7.00 g weight each were collected in localities around Naples, Italy. The animals were maintained in the laboratory under natural conditions of light (10^13 h) and temperature (ranging from 14 to 24‡C) and then decapitated under general anesthesia with ether vapors according to the institutional animal care and use committee. Temporal bones were removed and immersed in 0.9% NaCl. Otoconia were taken with a siliconized pulled Pasteur pipette under a stereomicroscope. The upper portion of mass of otoconia was taken with great care leaving a layer on the epithelium in order to be sure of taking only otoconia and not other cells and/or hairs contaminating the preparation. Otoconia from utricle and lagena were pooled while those from saccule were collected separately. Proteins from otoconia were extracted according to Piscopo et al. (2003). Protein extraction from papilla basilaris of two adult specimens of Gallus domesticus and from saccule, utricle and lagena (previously deprived of otoconia) of two adult specimens of P. sicula was performed according to Piscopo et al. (2003). Samples were analyzed using sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^ PAGE) to determine their protein content. 12.5% polyacrylamide gels were used with a 5% stacking gel. Laemmli’s Tris^glycine bu¡er was used for electrophoresis (Laemmli, 1970). Gels were run until the tracking dye had just left the gel and stained with silver nitrate (Merril et al., 1984).
Fig. 1. Scanning electron micrographs. A: Calcite otoconia (1400U). B: Aragonite otoconia (1500U).
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Fig. 2. A: SDS^PAGE analysis of: molecular weight standards (lane M); proteins extracted from P. sicula saccule otoconia (lane Ot.S); proteins extracted from P. sicula utricle plus lagena otoconia (lane Ot.U+L); proteins extracted from P. sicula saccule, utricle, lagena, ampulla, basilaris papilla epithelia (lanes S, U, L, A, P, respectively). B: Immunoblot assay on the same samples shown in A with monoclonal anti-calbindin D-28K; protein extracted from chicken basilaris papilla (+).
2.2. Western immunoblot Immediately after electrophoresis the gel was placed in transfer bu¡er (20% methanol, 0.025 mM Tris-base, 0.129 M glycine pH 8.5) and the nitrocellulose paper (Amersham 0.45 Wm pore) was placed in a separate dish containing fresh transfer bu¡er and four pieces of ¢lter paper. Filter was removed from the transfer bu¡er and placed on top of the gel. A sandwich of the nitrocellulose and gel was placed between the presoaked ¢lter
paper and placed in a Bio-Rad Mini transblot. The transfer took place for 2 h at 100 V constant at 4‡C. The electroblot was removed from the transfer apparatus and treated as follows: (1) 3% Nidina (not fat dry milk) in 0.1 M phosphate-bu¡ered saline (PBS) pH 7.2 (1% NaCl, 0.025% KCl, 0.144% Na2 HPO4 , 0.025% K2 HPO4 ) for 60 min at 37‡C, (2) primary antibody: monoclonal anti-calbindin D-28K (mouse IgG1 isotype) (Sigma, St. Louis, MO, USA) used at a dilution of 1/2500 in the same bu¡er for 2 h at 37‡C, (3) three
C Fig. 3. CLSM micrographs of P. sicula aragonite and calcite otoconia treated with anti-calbindin D-28K and rabbit FITC-conjugated antimouse. Aragonite otoconia decalci¢ed with: A2 : dH2 O at 37‡C for 24 h (1600U); B2 : dH2 O at 37‡C for 48 h (1600U); C, E: HCl 1 N at room temperature for 2 min (1100U); H2 : HCl 1 N at room temperature for 3 min (1300U). D, F, I: Three-dimensional reconstruction of otoconia shown in C, E, H2 , respectively. G2 : Calcite otoconia decalci¢ed with dH2 O at 37‡C for 24 h (600U); A1 , B1 , G1 , H1 : ICT images of crystals shown in A2 , B2 , G2 , H2 , respectively.
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washes with 0.1 M PBS and then secondary antibody : horseradish peroxidase rabbit anti-mouse IgG (Sigma, St. Louis, MO, USA) used at a dilution of 1/1000 for 1 h at 37‡C, (4) three washes with 0.1 M PBS, (5) addition of 10 ml of methanol containing 3 mg/ml 4-chloro-1naphthol, and then addition of 10 Wl of H2 O2 30 v/v. The ¢lter was kept on constant motion until the bands appeared and then washed with double-distilled water. 2.3. CSLM Otoconia were subjected to a mild (dH2 O at 37‡C for 12^72 h) or more heavy decalci¢cation (HCl 1 N at room temperature for 2^3 min) and then treated with monoclonal anti-calbindin D-28K (mouse IgG1 isotype) (Sigma, St. Louis, MO, USA) as primary antibody and rabbit £uorescein isothiocyanate (FITC)-conjugated anti-mouse as secondary antibody, each used at a dilution of 1:1000. Fluorescence observations were carried out by a confocal microscope (Leica TCSNT) with laser argon-krypton 7^5 mW multiline. Focal series of horizontal planes of sections were monitored for FITC using the 488 nm laser line and a bandpass 590/30-nm ¢lter. Preimmune sera, instead of speci¢c antisera, were used in the negative control. 2.4. Use of animals Samples captured with authorization of 1/06/2000 N. SCN/2D/2000/9213 del Ministero dell’Ambiente.
et al., 1988; Balsamo et al., 2000). Immunoreactivity was observed only in the sample containing the proteins extracted from otoconia of saccule (Fig. 2B, lane Ot.S) and in that containing the proteins extracted from saccule epithelium (Fig. 2B, lane S). These results have been con¢rmed by CLSM analyses that have also indicated the presence of calbindin D-28K not only on the surface but also in the core of aragonite crystals. In fact in these crystals after weak decalci¢cation (24^48 h in dH2 O at 37‡C), £uorescence on the surface of crystal is evident (Fig. 3A2 , B2 ) while in those decalci¢ed with HCl 1 N for 2^3 min at room temperature, £uorescence was found in the central portion of crystal (Fig. 3C, E, H2 ). Fig. 3A1 , B1 , G1 , H1 are the corresponding images, at transmitted light interference contrast (ICT) of crystals shown in Fig. 3A2 , B2 , G2 , H2 . The crystals in Fig. 3A1 and B1 show weak erosion while Fig. 3H1 shows only the protein core of crystal following to the stronger decalci¢cation. In the calcite crystals, either weakly or strongly decalci¢ed, £uorescence was not found (Fig. 3G2 ). A three-dimensional reconstruction obtained from the superimposition of 20 optical sections shows a typical aspect of otoconia after decalci¢cation (Fig. 3D, F, I). Fig. 3D, F show the aragonite crystals after decalci¢cation with HCl 1 N for 2 min. In this case the crystals are not yet completely decalci¢ed. Calbindin D-28K is evident in the protein core of aragonite crystal after complete decalci¢cation with HCl 1 N for 3 min (Fig. 3H2 ). All negative controls did not show £uorescence.
3. Results 4. Discussion In Fig. 2A is shown the SDS^PAGE of the proteins contained in the otoconia of saccule (lane Ot.S); in the otoconia of utricle plus lagena (lane Ot.U+L); and of the proteins extracted from P. sicula saccule, utricle, lagena, ampulla and papilla epithelia (lanes S, U, L, A and P, respectively), after otoconia were removed from the saccule, utricle and lagena. The samples show similar electrophoretic patterns and all seem to present a band of about 28 kDa. Since we have already shown that the organic matrix of P. sicula otoconia contains a protein of this molecular weight corresponding to calbindin D-28K, the same samples shown in Fig. 2A were rerun and the proteins transferred on a nitrocellulose ¢lter. The immunoblotting was performed with monoclonal anti-calbindin D-28K (Fig. 2B) in order to establish if calbindin could be present in all samples or it could be typical of a particular type of crystal and/or epithelia. The proteins extracted from chicken basilaris papilla were used as positive control (+), calbindin D-28K being one of its components (Oberholtzer
Calbindin D-28K belongs to the family of the CBPs. Special attention has been paid to these proteins because they modulate cellular Ca2þ level. Calbindin is a 28 kDa vitamin D-induced protein located in the peripheral vestibular system of various vertebrates (Dechesne et al., 1988a) where it could play a role in the electrophysiological functioning of the sensory cells. Expression of calbindin D-28K was seen in the hair cells and the vestibular ganglion on gestional day (GD) 19, in nerve ¢bers on GD23 and in otoconia on GD26 of the musk shrew (Karita et al., 1999). Sans et al. (1986) have suggested that the presence of calbindin D-28K may be associated with at least three important roles of Ca2þ in vestibular hair cells: transduction phenomena, contraction of sensory apex proteins and transmitter release. Numerous calbindin D-28K immunoreactive particles were found within the otolithic membrane on the sensory hair cells layer in the squirrel monkey (Usami et
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al., 1995). These ¢ndings may indicate that the Ca2þ transport mechanism is necessary in forming and maintaining otoconia. Recently we have demonstrated the presence of calbindin D-28K in the organic matrix of P. sicula otoconia (Piscopo et al., 2003) in line with Balsamo et al. (2000) who have found calbindin D28K immunoreactivity in the periphery and also in the central portion of some otoconia of chick embryos and adult animals indicating that otoconia are dynamic structures, which undergo turnover. Now we have evidenced by immunoblot assay with monoclonal anti-calbindin D-28K the presence of calbindin D-28K only in the aragonite otoconia and this result was con¢rmed also by CLSM analyses and indirectly also by the exclusive localization of this protein in the saccule epithelium (probable place of origin of the crystals). This ¢nding can be correlated to the more speci¢c function of calcareous reserve that aragonitic otoconia carry out in many classes of vertebrates, reptiles and amphibia (Guardabassi, 1952, 1953; Marmo et al., 1981, 1983a,b). This function of aragonite otoconia is likely due to the fact that Ca2þ of aragonite crystals is more easily mobilizable as demonstrated by several studies and also by scanning electron microscopy analyses that indicate evident signs of erosion of aragonite crystals, not present in calcite otoconia, in amphibia and reptiles (Marmo et al., 1986). It is possible to hypothesize that calbindin D-28K in those crystals could act as solubilizator of Ca2þ from the reserve deposits. In fact, calbindin D-28K is one of the Ca2þ bu¡ering proteins responsible for maintaining low levels of intracellular free Ca2þ (Slepecky and Ulfendhal, 1993). Usami et al., 1995, in fact, have suggested that Ca2þ in the endolymph may be taken up into organic substances in the otoconia via calbindin. Such a mechanism may start functioning during development. Thus, it is possible to hypothesize that calbindin D-28K can be involved both in the process of mineralization of otoconia, as demonstrated by the presence of this protein in the otoconia of chick embryo (Balsamo et al., 2000) and in their decalci¢cation in the case otoconia is functioning as calcareous reserve.
References Balsamo, G., Avallone, B., Del Genio, F., Marmo, F., 2000. Calci¢cation processes in the chick otoconia and calcium binding proteins: patterns of tetracycline incorporation and calbindin D-28K distribution. Hear. Res. 148, 1^8. Be'langer, L.F., 1960. Development, structure and composition of the otolithic organs of the rat. In: R.F. Sognnaes (Ed.), Calci¢cation in Biological Systems. American Association for the Advancement of Science, Washington, DC, pp. 151^162. Carlstro«m, D., 1963. A crystallographic study of vertebrate otolithis. Biol. Bull. 125, 421^463.
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organic material of otoconial complexes, including its amino acid and carbohydrate composition. In: Auditory Biochemistry, pp. 351^365. Sans, A., Etchecopar, B., Bhehier, A., Thomasset, M., 1986. Immunocytochemical detection of vitamin D-dependent calcium-binding protein (CaBP-28K) in the vestibular hair cells of the cat. Brain Res. 364, 190^194. Slepecky, M.B., Ulfendhal, M., 1993. Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti. Hear. Res. 70, 73^84. Usami, S., Shinkawa, H., Inoue, Y., Kanzaki, J., Anniko, M., 1995. Calbindin D-28K localization in the primate inner ear. ORL 57, 94^99. Veenho¡, V.B., 1969. The Development of Statoconia in Mice. Akademie van Wetenschappen, Afdeling Natuurkunde 2/58, No. 4. N.V. North-Holland, Amsterdam. Wislocki, G.B., Ladman, A.G., 1955. Selective and histochemical staining of the otolithic membranes cupulae and tectorial membrane of the inner ear. J. Anat. Lond. 89, 3^12.
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