Real-time bioluminescence imaging of a protein secretory pathway in living mammalian cells using Gaussia luciferase.
Using photon counting and charge-coupled device (CCD) cameras, we have applied the method of real-time bioluminescence imaging to investigate protein trafficking in mammalian cells. In the living cells of Chinese hamster ovary and PC12D cells, exocytotic secretion of protein and protein targeting on the cell surface were visualized using the secreted Gaussia luciferase (GLase) as a reporter protein in a minute. After incubation of the cells with luciferin (coelenterazine) for 10min, luciferin was imported into the cells and the vesicle transport network in the cells could be shown by luminescence images of GLase activity. Further, we demonstrate that GLase with a heterologous signal peptide sequence is targeted to the cell surface in neuronally differentiated PC12D cells and luminescence signals could be detected in a few seconds.
Suzuki
Takahiro
T
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Usuda
Shinsuke
S
Ichinose
Hiroshi
H
Inouye
Satoshi
S
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D015870:Gene Expression; D008156:Luciferases / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors