title: Single-molecule imaging of BMP4 dimerization on human periodontal ligament cells.
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.
authors.affiliation: Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University and National Taiwan University Hospital, No.1, Changde St., Jhongjheng District, Taipei 100, Taiwan, ROC.
authors.forename: H-W
authors.initials: HW
authors.lastname: Mi
authors.forename: M-C
authors.initials: MC
authors.lastname: Lee
authors.forename: Y-C
authors.initials: YC
authors.lastname: Chiang
authors.forename: L-P
authors.initials: LP
authors.lastname: Chow
authors.forename: C-P
authors.initials: CP
authors.lastname: Lin
mesh_terms: D055415:Bone Morphogenetic Protein 4 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002454:Cell Differentiation; D002462:Cell Membrane; D002478:Cells, Cultured; D003080:Cold Temperature; D019281:Dimerization; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D010513:Periodontal Ligament / Q000166:cytology* / Q000378:metabolism; D011485:Protein Binding; D045663:Quantum Dots; D011993:Recombinant Fusion Proteins
chemical_list: D055415:Bone Morphogenetic Protein 4; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins