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Golgi Labeling with Green Fluorescent Protein
- The Golgi complex has been labeled in living cells using four GFP chimeras:
mannosidase II-GFP, galactosyltransferase-GFP, KDEL receptor-GFP and KDEL
receptor mutant-GFP. When expressed transiently in HeLa cells, all of the
chimeras localized to the Golgi complex and co-localized with antibodies
to native Golgi proteins.
- Diffusional mobility of these chimeras was investigated using FRAP
(Fluorescence Recovery After Photobleaching). Galtase-GFP fluorescence
recovered from photobleaching by about 1 minute (Figure
2, 316K movie) (images collected every 4 sec). Diffusion coefficients
for the chimeras ranged from 3-5 x 10-9
Thus, the chimeras diffuse rapidly and freely in Golgi membranes, suggesting
that targeting and retention of Golgi proteins does not depend on immobilization.
- To investigate membrane continuity, a new variant of FRAP, called FLIP
(Fluorescence Loss In Photobleaching) was used. A small region of the Golgi
was repetitively bleached for 30 sec at a time. Between the bleaching periods,
the cell was imaged with low intensity light. Even though the area of bleaching
was small, the entire Golgi complex was darkened within 360 sec (Figure 3B, 868K movie). This is strong evidence that all
of the Golgi membranes are continuous with the membranes within the irradiated
region. In some cells, isolated Golgi elements remained bright during FLIP,
suggesting they were not continuous with the Golgi membranes being bleached
- FLIP also was used to investigate the mobility of the Galtase-GFP redistributed
into the ER by BFA (Figure 3C, 705K movie). Most
of ER-associated fluorescence was lost by 480 sec of repeated bleaching
of a small region of the ER. Thus, the GFP-chimeras are free to diffuse
throughout ER and all membranes of this organelle are interconnected.
These results reveal that Golgi proteins diffuse rapidly and freely within
Golgi membranes, as well as ER membranes. The FLIP experiments dramatically
demonstrate the continuity of each of these compartments.
Nelson Cole, CBMB, NICHD, NIH, Bethesda, MD firstname.lastname@example.org
Caroline Smith, NINDS, NIH, Bethesda, MD email@example.com
Noah Sciaky, CBMB, NICHD, NIH, Bethesda, MD, current address: National
Jewish Hospital, Denver, CO firstname.lastname@example.org
Mark Terasaki, Dept Physiology, University of Connecticut Health Center,
Farmington, CT email@example.com,
Michael Edidin, Johns Hopkins University Edidin@jhu.edu
Jennifer Lippincott-Schwartz, CBMB, NICHD, NIH, Bethesda, MD firstname.lastname@example.org.
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