Granulogenesis in non-neuroendocrine COS-7 cells induced by EGFP-tagged chromogranin A gene transfection: identical and distinct distribution of CgA and EGFP.
We examined whether an enhanced green fluorescent protein (EGFP)-tagged chromogranin A (CgA) gene construct could serve as a marker protein to follow the synthesis of CgA and the process of granulogenesis in non-neuroendocrine (NE) cells. We transfected a CgA-EGFP expression vector into non-NE COS-7 cells and investigated the localization of a chimeric CgA-EGFP protein using confocal laser scanning microscopy (CLSM). The fluorescent signal of CgA-EGFP was distributed granularly in the cytoplasm. An immunocytochemical study using anti-CgA antibody with a quantum dot (Qd)525 shows colocalization of fluorescent signal of chimeric CgA-EGFP and CgA-Qd525 signals in granular structures, particularly at the periphery of the cytoplasm. We interpreted granules that were immunoreactive to CgA in electron micrographs as secretory. Spectral analysis of EGFP fluorescence revealed distinct EGFP signals without CgA colocalization. This is the first report to show that a granular structure can be induced by transfecting the EGFP-tagged human CgA gene into non-NE cells. The EGFP-tagged CgA gene could be a useful tool to investigate processes of the regulatory pathway. A more precise analysis of the fluorescence signal of EGFP by combination with the Qd system or by spectral analysis with CLSM can provide insight into biological phenomena.
Inomoto
Chie
C
Dept of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan. cisophia@is.icc.u-tokai.ac.jp
Umemura
Shinobu
S
Egashira
Noboru
N
Minematsu
Takeo
T
Takekoshi
Susumu
S
Itoh
Yoshiko
Y
Itoh
Johbu
J
Taupenot
Laurent
L
O'Connor
Daniel T
DT
Osamura
R Yoshiyuki
RY
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D053379:Chromogranin A / Q000096:biosynthesis* / Q000235:genetics; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D053379:Chromogranin A; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins