Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Polish Academy of Sciences

In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos-2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos-2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na(+)/K(+) ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis.

Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast-like Saos-2 cells.

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC-ESI-MS/MS and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions.

Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells.

Nucleoside diphosphate kinases (NDPKs) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among the NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers, and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process: first, initiation by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. The ability of NDPK-B to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity Decrease and Changes in Lipid Lateral Organization and Protein Cluster Formation.

Members of Rho-associated coiled-coil kinases (ROCKs) are effectors of Rho family of small GTPases. ROCKs have multiple functions that include regulation of cellular contraction and polarity, adhesion, motility, proliferation, apoptosis, differentiation, maturation and remodeling of the extracellular matrix (ECM).

Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization.

Bone cells control initial steps of mineralization by producing extracellular matrix (ECM) proteins and releasing vesicles that trigger apatite nucleation. Using transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) we compared the quality of minerals in vesicles produced by two distinct human cell lines: fetal osteoblastic hFOB 1.19 and osteosarcoma Saos-2. Both cell lines, subjected to osteogenic medium with ascorbic acid (AA) and β-glycerophosphate (β-GP), undergo the entire osteoblastic differentiation program from proliferation to mineralization, produce the ECM and spontaneously release vesicles. We observed that Saos-2 cells mineralized better than hFOB 1.19, as probed by Alizarin Red-S (AR-S) staining, tissue nonspecific alkaline phosphatase (TNAP) activity and by analyzing the composition of minerals in vesicles. Vesicles released from Saos-2 cells contained and were surrounded by more minerals than vesicles released from hFOB 1.19. In addition, there were more F and Cl substituted apatites in vesicles from hFOB 1.19 than in those from Saos-2 cells as determined by ion ratios. Saos-2 and h-FOB 1.19 cells revealed distinct mineralization profiles, indicating that the process of mineralization may proceed differently in various types of cells. Our findings suggest that TNAP activity is correlated with the relative proportions of mineral-filled vesicles and mineral-surrounded vesicles. The origin of vesicles and their properties predetermine the onset of mineralization at the cellular level.

Characteristics of minerals in vesicles produced by human osteoblasts hFOB 1.19 and osteosarcoma Saos-2 cells stimulated for mineralization.

The process of physiological mineralization that occurs during bone ossification is a tightly regulated cascade of molecular events leading to formation of bony skeleton. Its deregulation associated with aging leads to pathological mineralization not only in osseous but also in soft tissues. Recent discoveries in the field of bone biology indicate the participation of the Src family of tyrosine kinases as well as the Rho family of small GTPases in mineral formation. Cross-talk between these two signaling pathways is activated during an early step of the mineralization process, and leads to reorganization of the cytoskeleton of mineralization-competent cells and to matrix vesicles release. The understanding of mechanisms of the cross-talk between Src kinases and Rho small GTPases that regulate the mineralization process is crucial for the development of novel simply imaging techniques and therapeutic strategies in relation to pathological mineralization.

Cross-talk between Src kinases and Rho small GTPases regulates biomineralization and simplify imaging of the mineralization process.

Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization.

Agnieszka Strzelecka-Kiliszek

Laboratory of Lipid Biochemistry,
Nencki Institute of Experimental Biology

Polish Academy of Sciences

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Agnieszka Strzelecka-Kiliszek

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Lipid Biochemistry,Nencki Institute of Experimental Biology

Polish Academy of Sciences

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

Laboratory of Lipid Biochemistry,
Nencki Institute of Experimental Biology