Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

Jens Rietdorf; David J. Stephens; Anthony Squire; Jeremy Simpson; David T. Shima; Jean-Pierre Paccaud; Philippe I.H. Bastiaens; Rainer Pepperkok

doi:10.1117/12.384216

27 April 2000 Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

Jens Rietdorf, David J. Stephens, Anthony Squire, Jeremy Simpson, David T. Shima, Jean-Pierre Paccaud, Philippe I.H. Bastiaens, Rainer Pepperkok

Author Affiliations +

Proceedings Volume 3921, Optical Diagnostics of Living Cells III; (2000) https://doi.org/10.1117/12.384216
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

Abstract

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in selective packaging of anterograde cargo into coated transport vesicles budding from the ER. COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER. We have used multi spectral 3D imaging to visualize COPI and COPII behavior simultaneously with various GFP-tagged secretory markers in living cells. This shows that COPII and COPI act sequentially whereby COPI association with anterograde transport complexes is involved in microtubule-based transport and the en route segregation of ER recycling molecules from secretory cargo within TCS in transit to the Golgi complex. We have also investigated the possibility to discriminate spectrally GFP fusion proteins by fluorescence lifetime imaging. This shows that at least two, and possibly up to three GFP fusion proteins can be discriminated and localized in living cells using a single excitation wavelength and a single broad band emission filter.

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Jens Rietdorf, David J. Stephens, Anthony Squire, Jeremy Simpson, David T. Shima, Jean-Pierre Paccaud, Philippe I.H. Bastiaens, and Rainer Pepperkok "Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells", Proc. SPIE 3921, Optical Diagnostics of Living Cells III, (27 April 2000); https://doi.org/10.1117/12.384216

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KEYWORDS

Green fluorescent protein

Proteins

Luminescence

Fluorescence lifetime imaging

Microscopes

Image fusion

Stereoscopy

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