Movie images

The movie images shown here were acquired using equipment available in the MRC Cell Imaging Facility by George Banting's Research group.

In the technique of Fluorescence Recovery After Photobleaching (FRAP), a region of fluorescence within the cell is subjected to photobleaching (by increased illumination). The rate at which the photobleached area regains fluorescence is then monitored and, in the case where the fluorescent signal is provided by a suitably tagged membrane protein, provides an indication of the diffusional mobility of the protein in the plane of the lipid bilayer.
This is because the photobleached region regains a fluorescent signal by virtue of being repopulated by fluorescently tagged membrane proteins which have diffused in from the surrounding non-bleached area. In the technique of Fluorescence Loss In Photobleaching (FLIP), a region of fluorescence within the cell is subjected to repeated photobleaching (by increased illumination).
Over time, this will lead to a loss of the fluorescent signal from throughout the cell if all the fluorescent molecules can diffuse past the point of increased illumination. The rate at which fluorescence is lost from the entire cell is monitored and (as with FRAP), in the case where the fluorescent signal is provided by a suitably tagged membrane protein, provides an indication of the diffusional mobility of the protein in the plane of the lipid bilayer.
This series of movies shows the results of FRAP and FLIP analysis of GFP-tagged sialyl transferase (GFP-ST) (a type 1 integral membrane protein which is localised to the trans face of the Golgi stack in control cells) expressed in stably transfected chick embryo fibroblast (CEF) cells.

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Movie 1 - FRAP analysis of GFP-ST in CEF cells. The area which is subject to photobleaching is indicated by the box. Note the lack of recovery of fluorescence in the photbleached area, indicating limited diffusional mobility of this construct in the plane of the lipid bilayer when in the trans Golgi. (Movie represents 10 minutes of imaging).

Movie 2 - FLIP analysis of GFP-ST in CEF cells. The area which is subject to repeated photobleaching is indicated by the box. Note that despite repeated photobleaching of one area of the cell, there is not a complete loss of fluorescence. This indicates limited diffusional mobility of the GFP-ST construct in the plane of the lipid bilayer when in the trans Golgi. (Movie represents 20 minutes of imaging)

Movie 3 - FRAP analysis of GFP-ST in BFA-treated CEF cells. Incubation of cells in the presence of brefeldin A (BFA) leads to a redistribution of Golgi localised membrane proteins into the ER membrane. So, in this case, at the start of the movie GFP-ST can bee seen to be present throughout the ER. The area which is subject to photobleaching is indicated by the box. Note the rapid recovery of fluorescence in the photobleached area (contrasting markedly with the events shown in movie 1), indicating rapid diffusional mobility of this construct in the plane of the lipid bilayer when in the ER. (Movie represents 5 minutes of imaging)

Movie 4 - FLIP analysis of GFP-ST in BFA-treated CEF cells. Incubation of cells in the presence of brefeledin A (BFA) leads to a redistribution of Golgi localised membrane proteins into the ER membrane. So, in this case, at the start of the movie GFP-ST can bee seen to be present throughout the ER. The area which is subject to repeated photobleaching is indicated by the box. Note the complete loss of fluorescence from the cell (contrasting markedly with the events shown in movie 2), indicating rapid diffusional mobility of this construct in the plane of the lipid bilayer when in the ER. (Movie represents 10 minutes of imaging)

Movie 5 - This movie represents a 3D reconstruction of the the trans Golgi network (TGN) within a living cell. The image was generated from a stack of confocal images of the TGN collected from a stably transfected rat NRK cel expressing GFP-tagged TGN38 (TGN38 is a type 1 integral membrane protein which is predominantly localised to the TGN).

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