Abstract
Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca2+]i) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca2+]i. Cryomethods, quick-freezing and freeze-drying, and OsO4 vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.
Similar content being viewed by others
References
Abramov AY, Duchen MR (2003) Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca(2+) ionophore on mitochondria in intact cells. Cell Calcium 33:101–112
Ahn C C, Krivanek OL (1983) EELS Atlas, Gatan, Warrendale
Anderson HA (1995) Molecular biology of matrix vesicles. Clin Orthop Relat Res 314:266–280
Beckers ALD, Gelsema ES, De Bruijn WC, Cleton-Soeteman, Van Eijk UG (1996) Quantitative electron spectroscopic imaging in bio-medicine: evaluation and application. J Microsc 183:78–88
Bordat C, Bouet O, Cournot G (1998) Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging. Histochem Cell Biol 109:167–174
Buchanan RA, Leapman RD, O’Connell MF, Reese TS, Andrews SB (1993) Quantitative scanning transmission electron microscopy of ultrathin cryosections: subcellular organelles in rapidly frozen liver and cerebellar cortex. J Struct Biol 110:244–255
Collins TJ, Berridge MJ, Lipp P, Bootman MD (2002) Mitochondria are morphologically and functionally heterogeneous within cells. EMBO J 21:1616–1627
De Buijn WC, Sorber CWJ, Gelsema ES, Beckers ALD, Jonkind JF (1993) Energy filtering transmission electron microscopy of biological specimens. Scanning Microsc 7:693–709
Edelmann L, Ruf A (1996) Freeze-dried human leukocytes stabilized with uranyl acetate during low temperature embedding or with OsO4 vapor after embedding. Scanning Microsc Suppl 10:295–307
Egerton RF (1996) Electron energy loss spectroscopy in the electron microscope. Plenum, New York
Gomez P, Vereecke J, Himpens B (2001) Intra- and intercellular Ca2+-transient propagation in normal and high glucose solutions in ROS cells during mechanical stimulation. Cell Calcium 29:137–148
Grignon N, Halpern S, Jeusset J, Briançon C, Fragu P (1997) Localization of chemical elements and isotopes in the leaf of soybean (Glycine max) by SIMS microscopy: critical choice of sample preparation procedure. J Microsc 186:51–66
Grohovaz F, Bossi M, Pezzati R, Meldolesi J, Tarelli FT (1996) High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve–muscle preparation. Proc Natl Acad Sci U S A 93:4799–4803
Hisada A, Yoshida T, Kubota S, Nishizawa NK, Furuya M (2001) Technical advance: an automated device for cryofixation of specimens of electron microscopy using liquid helium. Plant Cell Physiol 42:885–893
Ho R, Feng J, Shao Z, Somlyo AP (1999) Calcium quantitation with a parallel electron energy loss spectroscopy/cooled charge-coupled device/200 keV system. Microscopy and Microanalysis 5:17–28
Hsu HH, Anderson HC (1996) Evidence of the presence of a specific ATPase responsible for ATP-initiated calcification by matrix vesicles isolated from cartilage and bone. J Biol Chem 271:26383–26388
Johnson K, Jung A, Murphy A, Andreyev A, Dykens J, Terkeltaub R (2000) Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization. Arthritis Rheum 43:1560–1570
Jorgensen NR, Henriksen Z, Brot C, Eriksen EF, Sorensen OH, Civitelli R, Steinberg TH (2000) Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms. J Bone Miner Res 15:1024–1032
Jouaville LS, Pinton P, Bastianutto C, Rutter GA, Rizzuto R (1999) Regulation of mitochondrial ATP synthesis by calcium: evidence for a long-term metabolic priming. Proc Natl Acad Sci U S A 96:13807–13812
Komarova SV, Ataullakhanov FI, Globus RK (2000) Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts. Am J Physiol Cell Physiol 279:C1220–C1229
Leapman RD (2003) Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging. J Microsc 10:5–15
Leapman RD, Sun SQ, Hunt JA, Andrews SB (1994) Biological electron energy loss spectroscopy in the field-emission scanning transmission electron microscope. Scanning Microsc Suppl 8:245–258
Lian JB, Stein GS, Canalis E, Robey PG, Bosley AL (1999) Bone formation: osteoblast lineage cells, growth factors, matrix proteins, and the mineralization process. In: Favus MJ (ed) Primer on the metabolic bone diseases and disorders of mineral metabolism. Lippincott and Wilkins, Philadelphia, pp 20–29
Lieberherr M (1987) Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts. J Biol Chem 262:13168–13173
Lieberherr M, Grosse B, Kachkache M, Balsan S (1993) Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors. J Bone Miner Res 8:1365–1376
Monteith GR, Blaustein MP (1999) Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ. Am J Physiol 276:C1193–1204
Ottensmeyer FP (1984) Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope. J Ultrastruct Res 88:121–134
Pezzati R, Grohovaz F (1999) The frog neuromuscular junction revisited after quick-freezing–freeze-drying: ultrastructure, immunogold labelling and high resolution calcium mapping. Philos Trans R Soc Lond B Biol Sci 354:373–378
Pezzati R, Bossi M, Podini P, Meldolesi J, Grohovaz F (1997) High-resolution calcium mapping of the endoplasmic reticulum–Golgi–exocytic membrane system. Electron energy loss imaging analysis of quick frozen–freeze dried PC12 cells. Mol Biol Cell 8:1501–1512
Pezzati R, Meldolesi J, Grohovaz F (2001) Ultra rapid calcium events in electrically stimulated frog nerve terminals. Biochem Biophys Res Commun 285:724–727
Pivovarova NB, Hongpaisan J, Andrews SB, Friel DD (1999) Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics. J Neurosci 19:6372–6384
Pozzan T, Rizzuto R (2000) The renaissance of mitochondrial calcium transport. Eur J Biochem 267:5269–5273
Pozzo-Miller LD, Pivovarova NB, Leapman RD, Buchanan RA, Reese TS, Andrews SB (1997) Activity-dependent calcium sequestration in dendrites of hippocampal neurons in brain slices. J Neurosci 17:8729–8738
Reimer L, Zepke U, Moesch J, Schulze-Hillert S, Ross-Messmer M, Probst W, Weimer E (1992) EELS spectroscopy. Zeiss, Oberkochen
Rizzuto R, Brini M, Murgia M, Pozzan T (1993) Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262:744–747
Rizzuto R, Bastianutto C, Brini M, Murgia M, Pozzan T (1994) Mitochondrial Ca2+ homeostasis in intact cells. J Cell Biol 126:1183–1194
Rizzuto R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Tuft RA, Pozzan T (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763–1766
Robey PG, Boskey AL (2003) Extracellular matrix and biomineralization of bone. In: Favus MJ (ed) Primer on the metabolic bone diseases and disorders of mineral metabolism. Am Soc Bone Miner Res, Washington DC, pp 38–46
Rutter GA, Rizzuto R (2000) Regulation of mitochondrial metabolism by ER Ca2+ release: an intimate connection. Trends Biochem Sci 25:215–221
Shi SS, Andrews SB, Leapman RD (1996) Thickness measurement of hydrated and dehydrated cryosections by EELS. Microsc Res Tech 33:241–250
Somlyo AP, Urbanics R, Vadasz G, Kovach AG, Somlyo AV (1985) Mitochondrial calcium and cellular electrolytes in brain cortex frozen in situ: electron probe analysis. Biochem Biophys Res Commun 132:1071–1078
Sorber CWJ, Katelaars GAM, Gelsema ES, Jonkind JF, De Bruijn WC (1991) Quantitative analysis of electron energy-loss spectra from ultrathin-sectioned biological material. I. Optimization of the backgroundfit with the use of bio-standards. J Microsc 162:23–42
Stegmann H, Wepf R, Schroder RR, Fink RH (1999) Quantification of total calcium in terminal cisternae of skinned muscle fibers by imaging electron energy-loss spectroscopy. J Muscle Res Cell Motil 20:505–515
Trump BF, Berezesky IK (1995) Calcium-mediated cell injury and cell death. FASEB J 9:219–228
Tsai JA, Larsson O, Kindmark H (1999) Spontaneous and stimulated transients in cytoplasmic free Ca2+ in normal human osteoblast-like cells: aspects of their regulation. Biochem Biophys Res Comm 263:206–212
Wang HJ, Guay G, Pogan L, Sauve R, Nabi IR (2000) Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum. J Cell Biol 150:1489–1498
Wong G, Cohn DV (1974) Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells. Nature 252:713–715
Yang YY, Egerton RF (1995) Tests of two alternative methods for measuring specimen thickness in a transmission electron microscope. Micron 26:1–5
Acknowledgements
We are grateful to Dr. O. Boué (GAIB, CNAM–Paris ) for assistance in developing methods for EELS and ESI acquisitions and analysis, and to Dr. P. Gounon (INSERM, U 452, Nice, France) for help in cryofixation techniques. We thank Dr. O. Parkes for correcting the English text.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bordat, C., Guerquin-Kern, JL., Lieberherr, M. et al. Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy. Histochem Cell Biol 121, 31–38 (2004). https://doi.org/10.1007/s00418-003-0601-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-003-0601-9