Single-molecule imaging of Bmp4 dimerization on human periodontal ligament cells
Journal
Journal of Dental Research
Journal Volume
90
Journal Issue
11
Pages
1318-1324
Date Issued
2011
Author(s)
Abstract
We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the "blinking" behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs.Abbreviations: BMP4, bone morphogenetic protein 4; BMPR, BMP receptor; EGFP, enhanced green fluorescent protein; hPDL cells, human periodontal ligament cells; QDs, quantum dots; TIRFM, total internal reflection fluorescence microscopy; 293 cells, human embryonic kidney cells; oDM, osteogenic differentiation medium; HcoI, type I collagen; ALP, alkaline phosphatase; BSP, bone sialoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ? 2011 International & American Associations for Dental Research.
Other Subjects
bone morphogenetic protein 4; enhanced green fluorescent protein; fluorescent dye; green fluorescent protein; hybrid protein; quantum dot; article; biosynthesis; cell culture; cell differentiation; cell membrane; cell strain HEK293; chemistry; cold; cytology; dimerization; flow cytometry; fluorescence microscopy; gene expression; genetics; human; metabolism; methodology; molecular imaging; periodontal ligament; protein binding; Bone Morphogenetic Protein 4; Cell Differentiation; Cell Membrane; Cells, Cultured; Cold Temperature; Dimerization; Flow Cytometry; Fluorescent Dyes; Gene Expression; Green Fluorescent Proteins; HEK293 Cells; Humans; Microscopy, Fluorescence; Molecular Imaging; Periodontal Ligament; Protein Binding; Quantum Dots; Recombinant Fusion Proteins
Type
journal article