Brefeldin A (BFA) is a fungal metabolite that reversibly blocks protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. This effect is accompanied by a distinctive morphological change, the reversible collapse of the Golgi apparatus. The disassembly of the Golgi apparatus is accompanied by its dramatic tubulation, forming a network extending throughout the cytoplasm. While the content of the Golgi apparatus is redistributed to the ER, the content of the trans-Golgi network (TGN) collapses in the vicinity of the MTOC. This video shows the effect of BFA in an NRK cell stably expressing GFP-GMx33a, a protein associated to the TGN. NRK cells stably expressing GFP-GMx33a and plated on 35-mm glass-bottom culture dishes (MatTek), were treated with 5 microgram/ml BFA and maintained in phenol red-free buffered medium at 37C by using an ASI 400 Air Stream stage Incubator (Nevtek, Burnsville, VA). After 10 minutes in the presence of BFA time-lapse fluorescence images were acquired with an Ultraview Confocal Scanner (Perkin Elmer-Cetus, Norwalk, CT) in a Nikon Eclipse TE2000-S inverted microscope (Nikon, Melville, NY) equipped with a PlanApo 100x oil immersion objective (NA 1.40; Nikon), and a 12-bit charge-coupled device (CCD) camera, ORCA (Hamamatsu, Bridgewater, NJ). Image capture and data acquisition were performed using Ultraview LCI software (Perkin Elmer-Cetus). Images were acquired in binning 2 x 2 modes at approximately 0.25-s intervals, during approximately 175 s. Sequence images were exported as single TIFF files, and processed with ImageJ 1.36b (Rasband, 1997, 2007; http://rsb.info.nih.gov/ij/). Lippincott-Schwartz J, Donaldson JG, Schweizer A, Berger EG, Hauri HP, Yuan LC, Klausner RD. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell [serial online]. 1990;60:821-36. Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner RD. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell [serial online]. 1991;67:601-16. Snyder CM, Mardones GA, Ladinsky MS, Howell KE. GMx33 associates with the trans-Golgi matrix in a dynamic manner and sorts within tubules exiting the Golgi. Mol Biol Cell [serial online]. 2006;17:511-24.
- Biological Process
- Mardones, Gonzalo A.
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