Volume 4 issue 11 November 2003
Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo
Clément Nizak, Silvia Martin-Lluesma, Sandrine Moutel, Aurélien Roux, Thomas E. Kreis, Bruno Goud and Franck Perez

Movie 1. Figure 5. Monitoring endogenous Giantin dynamics in vivo by intracellular expression of the TA10-xFP intrabody. A. TA10-GFP was expressed in HeLa cells which were fixed with 3% paraformaldehyde 24 hr later and processed for immunofluorescence using anti-Giantin and anti-α-Mannosidase II antibodies. TA10-GFP interacts with its target in vivo (a) and the GFP fluorescence colocalizes perfectly with the anti-Giantin staining (b) and outlines the α-Mannosidase II Golgi staining (c). The overlay of the three staining is shown in (d). Bar = 10 µm. B. Endogenous Giantin detected by TA10-YFP (a) outlines the Golgi stained with GalNacT2-CFP (b) in living HeLa cells. HeLa cells were co-transfected with TA10-YFP and GalNacT2-CFP and time-lapse-imaged on a confocal microscope (see Supplemental Movie).

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Movie 2. Figure 6. Behavior of endogenous Giantin in vivo upon BFA treatment. HeLa cells co-expressing the Golgi localized marker GalNacT2-CFP together with TA10-YFP were treated with BFA and immediately followed by confocal time-lapse imaging every 20 s. The initial image (00:00), acquired at a lower scanning rate and hence of better quality, shows that endogenous Giantin detected by TA10-YFP outlines the Golgi staining of GalNacT2-CFP. GalNacT2-CFP-positive tubes emanating from the Golgi at early time points (04:00) and moving towards the periphery are devoid of endogenous Giantin (arrowheads). Upon longer incubation (05:20 to 06:20), GalNacT2-CFP and endogenous Giantin simultaneously blink out (arrows) as Golgi membranes suddenly fuse with the ER (see Supplemental Movie). Time is indicated in MM:SS. Bar = 10 µm.

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Movie 3. Figure 7. Behavior of endogenous Giantin in vivo upon microtubule depolymerization. A. HeLa cells co-expressing the Golgi marker GalNacT2-CFP and TA10-YFP were treated with Nocodazole and immediately confocal-imaged every 30 s for 90 min. Golgi mini-stacks which appear at the cell periphery are labeled with GalNacT2-CFP, likely because this marker recycles through the ER and accumulates on scattered, de novo formed, Golgi mini-stacks (arrows). In the same conditions, endogenous Giantin detected by TA10-YFP does not localize on these mini-stacks (arrows) but rather remains localized on the "old Golgi" that slowly fragments upon Nocodazole addition (see Supplemental Movie). Time is indicated in HH:MM:SS. B. Nocodazole treatment was repeated on wild-type HeLa cells and analyzed by immunofluorescence of endogenous proteins after fixation in Methanol. After 90 min of treatment, endogenous GalT (a, d) and GM130 (e) label scattered Golgi mini-stacks present in the cell periphery (arrows). In the same conditions, endogenous Giantin (b) is only detected on the "old" Golgi that remained localized in a juxtanuclear region. Bars = 10 µm.

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