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1.
Fig. 2.

Fig. 2. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

Arf1[Q71L] expression inhibits mitotic Golgi breakdown. NRK cells were transfected with GalT-YFP (Left) or Arf1[Q71L] and GalT-YFP (Center), and mitotic stages were identified by using Hoechst 33342 (Right). Shown are representative mitotic states illustrating the distribution of Golgi membranes. Arf1[Q71L]-expressing cells were easily identifiable based on their unique Golgi morphology and lack of sensitivity to BFA. (Bar, 10 μm.)

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.
2.
Fig. 4.

Fig. 4. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

Arf1 dynamics in untreated, H89-treated, and Arf1[Q71L]-expressing cells. (A) Photobleaching the Golgi pool of Arf1-GFP in interphase and prophase cells and monitoring recovery of fluorescence. (B) An example of recoveries observed for one interphase cell and one prophase cell. The Golgi to cytoplasm ratio of Arf1-GFP was plotted through the course of the experiment. (C) Photobleaching the Golgi pool of Arf1-GFP in an H89-treated metaphase cell or an Arf1[Q71L]-GFP-expressing cell. In H89-treated cells, Arf1-GFP recovered rapidly upon photobleaching the Golgi pool of its fluorescence (Upper), whereas Arf1[Q71L]-GFP-expressing cells showed negligible recovery over the same time period (Lower). (D) Model for Arf1 inactivation during mitosis and the effects of H89 and Arf1[Q71L]. Recruitment of Arf1 to Golgi membranes is inhibited early in mitosis by a mechanism that is sensitive to H89. This leads to the accumulation of Arf1-GDP in the cytoplasm, the inability to recruit Arf1 effector proteins onto Golgi membranes, and the disassembly of the Golgi. These mitotic effects can be inhibited either by expression of Arf1[Q71L] or by H89 treatment. (Bar, 10 μm.)

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.
3.
Fig. 1.

Fig. 1. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

H89 treatment blocks Golgi fragmentation and dispersal in mitosis. (A) Confocal images of control and H89-treated synchronized NRK cells expressing GalT-YFP at different stages of mitosis. (Top and Middle) GalT-YFP distribution. (Bottom) Hoechst staining of DNA in the same H89-treated cell. H89 was added to synchronized cells ≈30-45 min before mitosis, when cells were in G2 as determined by the lack of DNA condensation and histone-H3 phosphorylation (not shown). (Bar, 5 μm.) (B) Silver-enhanced ImmunoGold labeling of GalT-YFP in a metaphase NRK cell treated with H89 showed an enrichment of gold particles in Golgi-like fragments. At higher magnification (Inset), gold particles were over stacked Golgi cisternae. (C) In a control metaphase cell, gold particles were distributed throughout the cell. At higher magnification (Inset), gold particles predominantly associated with tubular/vesicular profiles. (Bars, 5 μm at low magnification and 1 μm at high magnification.)

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.
4.
Fig. 5.

Fig. 5. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

Inhibiting Arf1 inactivation in mitotic cells causes defects in chromosome segregation and cytokinesis. (A) Synchronized NRK cells expressing histone 2B-CFP were either untreated (control), treated with H89 (50 μM) 30 - 45 min before mitotic entry (H89), or treated with BFA (5 μg/ml) 15 min before the addition of H89 (BFA+H89) while cells were in G2 phase. Chromosomes were imaged by confocal microscopy as cells exited mitosis. In H89-treated cells, chromosome pairs did not completely separate in telophase (arrows), whereas in cells treated with BFA to disassemble the Golgi before H89, no chromosome bridging was observed. (B) Cells were examined by bright-field microscopy to observe cytokinesis. Control cells (Left) and cells treated with BFA before H89 (Right) completed furrow ingression, whereas H89-treated cells (Center) did not form a contractile furrow. (C) Untransfected, Arf1[Q71L]-transfected or H89-treated mitotic cells were stained with Hoechst 33342 to visualize telophase chromosomes. Bright-field images were collected from each cell to assess cytokinetic furrow ingression.

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.
5.
Fig. 6.

Fig. 6. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

Inhibiting Arf1 inactivation blocks the mitotic dispersion of a wide variety of peripheral Golgi proteins. (A) Interphase cells treated without or with BFA (5 μg/ml, 30 min) and mitotic cells in metaphase treated with or without H89 (50 μM) were fixed and stained with antibodies against tankyrase 1 (3T3-L1), Sak1 (HeLa), Cullin-2 (EA.hy926), myosin IIA (NRK), ankyrin 195 (NRK), and εCOPI (NRK). (Bar, 5 μm.) (B) Table summarizing the effect of short and long BFA treatments on Golgi proteins. (C) Peripheral Golgi proteins start to disperse in prophase before Golgi membranes fragment. Interphase and prophase cells (staged with Hoechst 33342) were labeled for Golgi peripheral (myosin IIA, tankyrase, and COP1) and integral membrane (GalT) proteins. ROIs were drawn around the Golgi and the whole cell. The Golgi fluorescence for each protein was background subtracted, expressed as a fraction of the total cell fluorescence, and normalized to the interphase values for 10 cells.

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.
6.
Fig. 3.

Fig. 3. From: A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

Arf1 distribution in mitosis. (A) Confocal images of mitotic NRK cells expressing Arf1-GFP. (B) Diffusion coefficients, D, of Arf1-GFP and free GFP in metaphase cells (n = 10 for each) and of free GFP and 40-nm fluorescent beads in PBS measured by using fluorescence correlation spectroscopy (FCS). The estimated D for beads in metaphase cells was extrapolated from the D for beads in PBS by using the difference in D observed between GFP in PBS and GFP in a metaphase cell. (C) Individual synchronized NRK cells (n = 10) coexpressing Arf1-CFP and GalT-YFP were followed as they advanced through mitosis. A simple image-processing method involving two regions of interest (ROI) was used to quantify Golgi and non-Golgi pools of Arf1-CFP and GalT-YFP. One ROI was drawn around GalT-YFP-labeled structures and fragments (representing Golgi labeling), and the other ROI was drawn around the rest of the cell (representing non-Golgi labeling). During each stage of mitosis, the mean fluorescence intensity associated with the Golgi and non-Golgi ROIs was measured for both Arf1-CFP and GalT-YFP. The total Arf1-CFP fluorescence associated with the Golgi was expressed as a fraction of the total cellular fluorescence (i.e., the sum of the Golgi and non-Golgi contributions) and normalized to the initial interphase value. This procedure was repeated for GalT-YFP, and both were plotted. (D) Arf1-GFP distribution in H89-treated interphase and metaphase cells that were treated before entry into mitosis. (E) Arf1[Q71L]-GFP expression in interphase and metaphase cells. (Bar, 10 μm.)

Nihal Altan-Bonnet, et al. Proc Natl Acad Sci U S A. 2003 November 11;100(23):13314-13319.

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