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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 cm² sec^-1. 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 (movie, 826K).
  
• 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.

Caroline Smith, NINDS, NIH, Bethesda, MD clsmith@codon.nih.gov

Noah Sciaky, CBMB, NICHD, NIH, Bethesda, MD, current address: National Jewish Hospital, Denver, CO kupferlab@njc.org

Mark Terasaki, Dept Physiology, University of Connecticut Health Center, Farmington, CT terasaki@panda.uchc.edu, home page

Michael Edidin, Johns Hopkins University Edidin@jhu.edu

Jennifer Lippincott-Schwartz, CBMB, NICHD, NIH, Bethesda, MD jlippin@helix.nih.gov.

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