Articles by Hieng Chiong Tie in JoVE
Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass Hieng Chiong Tie1, Bing Chen1, Xiuping Sun1, Li Cheng2,3, Lei Lu1 1School of Biological Sciences, Nanyang Technological University, 2Bioinformatics Institute, 3School of Computing, National University of Singapore The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.
Other articles by Hieng Chiong Tie on PubMed
A Novel Imaging Method for Quantitative Golgi Localization Reveals Differential Intra-Golgi Trafficking of Secretory Cargoes Molecular Biology of the Cell. Mar, 2016 | Pubmed ID: 26764092 Cellular functions of the Golgi are determined by the unique distribution of its resident proteins. Currently, electron microscopy is required for the localization of a Golgi protein at the sub-Golgi level. We developed a quantitative sub-Golgi localization method based on centers of fluorescence masses of nocodazole-induced Golgi ministacks under conventional optical microscopy. Our method is rapid, convenient, and quantitative, and it yields a practical localization resolution of ∼ 30 nm. The method was validated by the previous electron microscopy data. We quantitatively studied the intra-Golgi trafficking of synchronized secretory membrane cargoes and directly demonstrated the cisternal progression of cargoes from the cis- to the trans-Golgi. Our data suggest that the constitutive efflux of secretory cargoes could be restricted at the Golgi stack, and the entry of the trans-Golgi network in secretory pathway could be signal dependent.
The Development of a Single Molecule Fluorescence Standard and Its Application in Estimating the Stoichiometry of the Nuclear Pore Complex Biochemical and Biophysical Research Communications. Sep, 2016 | Pubmed ID: 27613095 We report here an image-based method to quantify the stoichiometry of diffraction-limited sub-cellular protein complexes in vivo under spinning disk confocal microscopy. A GFP single molecule fluorescence standard was first established by immobilizing His-tagged GFP molecules onto the glass surface via nickel nitrilotriacetic acid functionalized polyethylene glycol. When endogenous nucleoporins were knocked down and replaced by the exogenously expressed and knockdown-resistant GFP-nucleoporins, the stoichiometry of the nucleoporin was estimated by the ratio of its fluorescence intensity to that of the GFP single molecules. Our measured stoichiometry of Nup35, Nup93, Nup133 and Nup88 is 23, 18, 14 and 9 and there are possibly16 copies of Nup107-160 complex per nuclear pore complex.