Single-molecule imaging of Bmp4 dimerization on human periodontal ligament cells

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.