Volume 5 issue 4 April 2004
Golgi inheritance under a block of anterograde and retrograde traffic
Clément Nizak^{1, 3}, Rachid Sougrat^{1, 2, 4}, Florence Jollivet¹, Alain Rambourg², Bruno Goud1 and Franck Perez¹

1 Institut Curie, CNRS UMR144, 26 rue d'Ulm 75248 Paris cedex 05 France
2 Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette, France.
3 present address: Rockefeller University, Center for studies in Physics and Biology, Living matter laboratory, box 34, 1230 York Avenue, 10021 New York, NY, USA.
4 present address: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, 18 library drive, Building 18T/Room 101, National Institutes of Health, Bethesda, MD 20892-5430, USA.

Correspondence should be addressed to F.P.:
Institut Curie, UMR144 CNRS, 26 rue d'Ulm 75248 Paris Cedex 05 France
Phone (33) 1 42 34 63 88
Fax (33) 1 42 34 63 82
franck.perez@curie.fr

Supplemental Movie 1. Movie corresponding to Fig. 1A.
Fig. 1. Analysis of BFA effects throughout the cell cycle.
A. Unsynchronized GalNacT2-GFP HeLa cells were treated with BFA or not (Ctrl) and imaged by time-lapse microscopy (time indicated in HH:MM). When BFA treatment was initiated in interphase, GalNacT2 was quickly relocated in the ER in 15 min (00:15) and remained so as cells progressed through mitosis. When BFA treatment was initiated in prometaphase, Golgi fragments did not disperse further (00:15), in contrast to their full dispersion in metaphase control cells (Ctrl, 01:15), and these fragments persisted instead throughout mitosis, aggregating into a BFA-resistant Golgi-like structure in telophase (00:45). When BFA treatment was initiated in metaphase, as the Golgi is the most dispersed (at 00:00 the GalNacT2-GFP signal is diffuse and only excluded from the metaphase plate), the same BFA-resistant Golgi-like structure formed in telophase, with an additional ER background (01:00). When BFA treatment was initiated as the Golgi had already started to reform (late anaphase/telophase), GalNacT2 was quickly relocated in the ER (00:15). Bar: 10 µm.

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Supplemental Movie 2. Movie corresponding to Fig. 1B.
B. GalNacT2-GFP HeLa cells were treated with BFA for 1 hour. Telophase cells containing a BFA-resistant Golgi-like structure or not were observed by time-lapse microscopy upon BFA washout (time indicated in HH:MM). The Golgi reformed in all cells, regardless of the presence (upper cell) or absence (lower cell) of the Golgi-like structure initially. When present, the Golgi-like structure intermixed with the reassembling Golgi (arrows), as de novo formed GalNacT2-GFP-positive structures converged towards this site. Bar: 10 µm.

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Supplemental Movie 3. Movie corresponding to Fig. 6B.
Fig. 6. The Golgi-like BFA-resistant structure is distinct from ER exit sites and is not connected to the ER. B. Mitotic GalNacT2-GFP HeLa cells were collected from non-synchronized cell populations, BFA was then immediately added or not, and cells were allowed to reach telophase. As illustrated in Fig. 1, if BFA was added during anaphase/telophase, GalNacT2-GFP was relocated in the ER (middle panel). In these conditions, a 15 min-FLIP of the ER pool of GalNacT2-GFP within the designated region of interest (ROI) led to a 95% reduction in total cell fluorescence (the second daughter cell is not photobleached and provides an internal control). In contrast, the BFA resistant Golgi-like structure observed in cells treated during metaphase (bottom panel), as well as the Golgi in control, non-treated cells (upper panel), were not bleached upon photobleaching of the ER by the same 15 min-FLIP procedure. Therefore, the Golgi-like structure is not directly connected to the ER, like the normal Golgi in control, non-treated cells. Bar: 10 µm.

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Supplemental Movie 4. Movie corresponding to Fig. 7B.
Fig. 7. The BFA-resistant Golgi-like structure forms in the absence of anterograde transport towards the Golgi.
B. The Golgi-like structure or the Golgi area was photobleached in one of the 2 daughter cells in cells treated with BFA in prometaphase/metaphase (lower panel) or non-treated cells (upper panel) respectively. The 2 min-photobleaching procedure ensured that recovery would not be due to lateral diffusion within connected membranes. The fluorescence recovery was then monitored for 30 min. No recovery was observed in BFA-treated cells while normal recovery, typical of ER-Golgi anterograde transport, was observed in control cells. Bar: 10 µm.

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Supplemental Movie 5. Movie corresponding to an additional experiment. The same FRAP experiment as in Fig. 7B was repeated with a bleached region corresponding to only one of the 2 BFA-resistant Golgi-like pools present in each daughter cells (centrosome- or midbody-associated).

This movie shows a cell in telophase that has formed a BFA-resitant Golgi-like structure. In the lower daughter cell, a portion of a Golgi-like structure was bleached, and recovery occurred within 3/5 minutes. In the upper daughter cell, one of the two Golgi-like structures, associated with the midbody, was bleached, and rapid recovery was also observed. Movement of GalNacT2-GFP-positive membranes coming from the juxtanuclear Golgi-like structure towards the bleached region was tracked.
Very intense membrane transport occurs between the two BFA-resistant structures (centrosome- and midbody-associated in the upper daughter cell), and the recovery after photobleaching is mainly due to transport of membrane between these two structures. Such a transport is also observed between the two microtubule minus end-associated Golgi pools in control, non-treated, cells. This result highlights the dynamics of BFA-resistant membranes, presumably along microtubules.

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Supplemental Movie 6. Movie corresponding to an additional in vivo experiment. The same FLIP protocol as in Fig. 6B was applied to non-treated metaphase GalNacT2-GFP HeLa cells, that were then observed and continuously photobleached as they progressed through mitosis.
In metaphase, the GalNacT2-GFP staining is essentially diffuse, but small structures are visible. In the course of the FLIP experiment, the diffuse fluorescence signal is reduced while the fluorescent structures become more contrasted, and aggregate to form the Golgi in anaphase/telophase. This shows that small Golgi structures naturally exist in metaphase HeLa cells, and that the Golgi is reformed at least in part from these coalescing structures. We propose that the Golgi-like fragments that become frozen and aggregate into the BFA-resistant structure observed in telophase in cells treated in metaphase (Fig. 1, 2) are originated from naturally existing metaphase Golgi fragments that normally fuse together as the Golgi reforms in non-treated cells.

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Supplemental Movie 7. Movie corresponding to an additional in vivo experiment.
The same experiment as in Fig.1A was repeated but this time BFA was added on non-synchronized GalNacT2-GFP HeLa cells when a cell in the field, which was in interphase at the beginning of the recording, entered mitosis and reached metaphase. The Golgi-like structure forms upon mitosis exit in this cell while Golgi blink-out occurs in all other cells. On this type of recordings, we have quantified the fluorescence signal associated with the Golgi in interphase (just before mitosis entry) and the signal associated with the Golgi-like structure in the same cell. We find that 60+/-7% of the total cell fluorescence is associated with the Golgi in interphase, and 30+/-8% with the Golgi-like structure in telophase. This means that the Golgi-like-structure contains 50+/-15% of the signal originally present in the Golgi before mitosis when BFA is added in metaphase. When BFA is added in prometaphase, we found that 43+/-5% of total cell fluorescence was present in the Golgi-like structure in telophase. So 70+/-12% of the signal originally associated with the Golgi is contained in the Golgi-like structure when BFA is added in prometaphase.

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