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ER to Golgi traffic visualized in living cells

John F. Presley. Nelson B. Cole, Trina A. Schroer, Koret Hirschberg, Kristien Zaal and Jennifer Lippincott-Schwartz

Nature,Volume 389, Pages 81-85, 1997

GFP was attached to the C-terminus of VSVG, a temperature sensitive viral glycoprotein which moves through the secretory pathway to the cell surface. Transport intermediates carrying VSVG-GFP from the ER to the Golgi complex were studied upon shift from 40°C (where VSVG-GFP is retained in the ER) or 15°C (where it accumulates in pre-Golgi structures) to 32°C (where it moves to the Golgi before transport to the cell surface).

Fig. 1B. Movie. Clustering of pre-Golgi structures labeled with VSVG-GFP upon shift from 15°C to 32°C. Images were collected every 3.5 sec for 9 min. The data reveal pre-Golgi structures translocate in a stop - and - go fashion inward toward the Golgi complex at rates of up to 1.4 µm s^-1. Pre- Golgi structures were irregularly shaped and frequently stretched into long tubules as they moved toward the center of the cell.

Fig. 3A-C. Movie. Formation and translocation of pre-Golgi structures containing VSVG-GFP in cells shifted from 40°C to 32°C. Images were collected at 8.6 sec intervals for 9 min. The first image is a cell approximately 5 min after shift to 32°C. The data show that pre-Golgi structures form at widely dispersed sites from the reticular ER and stay at such sites for only brief periods before translocating into the Golgi region.

Fig. 3E. Movie. Fate of pre-Golgi structures upon bleaching the Golgi region. The Golgi area was emptied of flourescence by bleaching a rectangular box with a high energy laser scan of the confocal microscope. Low intensity illumination was then used to image movement of pre-Golgi structures into the bleached area. Note that pre-Golgi structures quickly moved into the Golgi region and transferred flourescent material to the Golgi complex. Importantly, these pre-Golgi structures appeared to be the only source for transfer of flourescence to the Golgi complex. Other examples of this experiment can be seen in the movies Fig. 3E' and Fig. 3E''.

Fig. 4E. Movie. Effect of nocodazole on clustering of pre-Golgi structures. Nocodazole depolymerizes microtubules.

Fig. 4F. Movie. Clustering of pre-Golgi styructures upon nocodazole washout.

Fig. 4G. Movie. Effect of dynamitin overexpression on clustering of pre-Golgi structures. Dynamitin is a molecular motoer that moves on microtubule tracks.

E-mail addresses of authors:

John Presley, CBMB, NICHD, NIH, Bethesda, Md jpresley@helix.nih.gov

Nelson Cole, CBMB, NICHD, NIH, Bethesda, Md nbcole@box-n.nih.gov

Trina Schroer, Dept.Biology, The Johns Hopkins Univ., Baltimore, Md schroer@jhunix.hcf.jhu.edu

Koret Hirschberg, CBMB, NICHD, NIH, Bethesda, Md kotyh@box-k.nih.gov

Kristien Zaal, CBMB, NICHD, NIH, Bethesda, Md kamzaal@box-k.nih.gov

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

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