Cell Staining: Fluorescent Labelling of the Golgi Apparatus

Abstract

Immunofluorescence has been the primary method for labelling the Golgi apparatus for light microscopic observation in fixed cells. The Golgi apparatus was initially labelled in living cells using the fluorescent ceramide analogue N‐6[7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl] aminocaproyl sphingosine or by microinjection of fluorescently conjugated antibodies to exposed Golgi epitopes. With the common availability of the green fluorescent protein (GFP) and advanced fluorescence microscopes including confocal microscopes, imaging of the Golgi apparatus in living cells is now a major experimental approach. Time‐lapse imaging of living cells has also been combined with photobleach techniques in order to study the dynamics of transient protein association with the Golgi apparatus. Similar live‐cell imaging approaches have been used to study the dynamics of the cargo transport through the exocytic pathway. Recent advances in microscopy have allowed bypassing the diffraction limit which has traditionally limited image resolution. These new methods may be applied to fluorescence imaging of the Golgi apparatus, although they are not easily applied to living cells at present.

Key Concepts

  • Immunofluorescence can be used to visualise proteins in the Golgi apparatus or its subdomains.
  • Live‐cell visualisation of the Golgi apparatus is possible with GFP‐tagged proteins.
  • Fluorescence images can be quantitated in a rigorous way.
  • Photobleaching and photoactivation techniques permit visualisation of protein dynamics and flux through the Golgi apparatus in living cells.
  • Super‐resolution imaging promises a new generation of imaging approaches to the Golgi apparatus.

Keywords: Golgi apparatus; exocytosis; immunofluorescence; vital staining; GFP; photobleach; fluorescence microscopy; super‐resolution; PALM; STORM

Figure 1. Normal rat kidney (NRK) cells stained with (a) rabbit antibody directed against the cis‐Golgi marker p115 and a Cy5‐tagged secondary antibody transfected with (b) GFP‐tagged Rab33b, a cytosolic GTPase localising to the medial‐Golgi, and (c) the TGN marker TGN38 visualised with a mouse monoclonal antibody and a Cy3‐tagged secondary antibody localising to the trans‐cisternae of the Golgi and the trans‐Golgi network. (d) Merged images show that the localisation of the proteins does not overlap completely. P115 (blue), Rab33b (green) and TGN38 (red).
Figure 2. COS7 cells transfected with GFP‐p58/ERGIC53 which localises to the endoplasmic reticulum, Golgi apparatus and intermediate compartment. (a) Photobleach sequence described in the text. The region containing the Golgi apparatus and Golgi‐associated intermediate compartment was bleached and recovery visualised as indicated. (b) Quantitation of fluorescence recovery in the Golgi region in a photobleach experiment. Fluorescence in the Golgi region is normalised to total cell fluorescence. The initial point immediately precedes the bleach. Note that in this experiment a plateau is reached below the initial prebleach level, indicating some immobile fraction.
Figure 3. Example of STORM super‐resolution visualisation of the Golgi region of a Chinese Hamster Ovary (CHO) cell expressing GFP‐ϵCOP, which localises to coated pits and vesicles 50–60 nm in size. As these structures are densely packed on the Golgi apparatus, they cannot be individually resolved with a standard fluorescence microscope, although the pits and vesicles are visible by electron microscopy. GFP‐ϵCOP has been stained with an anti‐GFP rabbit primary antibody and a Cy3‐tagged secondary antibody. A special buffer has rendered most Cy3 molecules non‐fluorescent, but at any one time, a small random subset will be fluorescent. These molecules can be visualised individually. (a, b) Images taken at two different times using a specialised commercial system (Zeiss Elyra) capable of visualising fluorescence from individual molecules. Because of the diffraction of light, the fluorescence from each molecule appears to cover several hundred nanometers (fluorescent spots in (a) and (b)), yet because the fluorescent molecules are sparse, they can be individually localised to very high precision by localising the centre of each spot. The entire image sequence (1000 total frames) was taken over sufficient time that a large fraction of the molecules could be individually localised. (c) The superimposed localisation of all identified molecules at higher resolution (∼30 nm) than possible in a diffraction‐limited image. At this resolution, it is clear that most of the GFP‐ϵCOP is in small discrete structures which may be individual coated vesicles.
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Further Reading

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Dejgaard, Selma Y, Dejgaard, Kurt, and Presley, John F(May 2015) Cell Staining: Fluorescent Labelling of the Golgi Apparatus. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002633.pub3]