Mi H.-W.Lee M.-C.YU-CHIH CHIANGLU-PING CHOWCHUN-PIN LIN2020-01-222020-01-22201100220345https://www.scopus.com/inward/record.uri?eid=2-s2.0-80053632330&doi=10.1177%2f0022034511418340&partnerID=40&md5=616d4d64c323df481ffa8ac3bba36347https://scholars.lib.ntu.edu.tw/handle/123456789/452418We 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.[SDGs]SDG3[SDGs]SDG15bone 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 Proteinsjournal article10.1177/00220345114183402-s2.0-80053632330