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First published online 22 August 2006
doi: 10.1242/jcs.03146

Journal of Cell Science 119, 3764-3775 (2006)
Published by The Company of Biologists 2006
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Golgi structural stability and biogenesis depend on associated PKA activity

Eloy Bejarano*, Margarita Cabrera*, Lucia Vega, Josefina Hidalgo and Angel Velasco{ddagger}

Department of Cell Biology, Faculty of Biology, University of Seville, Avd. Reina Mercedes s/n, 41012 Seville, Spain


Figure 1
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  		Fig. 1. Kinetics of RII{alpha} binding to and dissociation from the Golgi. Cells stably expressing RII{alpha}-GFP were photobleached with a high-intensity laser light and then the recovery of fluorescence was monitored by taking images at the indicated time points (in minutes). (A) The Golgi complex (outlined region) was subjected to a single bleach and recovery was analyzed by FRAP. Note that the centrosome was excluded from the bleached area. (B) The cytoplasm (area between the two encircled structures) was bleached repeatedly (every 4 minutes) to analyze RII{alpha}-GFP loss from the Golgi by FLIP. (C) Time-course of the evolution of Golgi-associated fluorescence during both FRAP and FLIP. Values are the mean ± s.e.m. of three different determinations.

 

Figure 2
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  		Fig. 2. RII{alpha} depletion by siRNA treatment. HeLa cells were incubated (+) or not (-) for 72 hours with an siRNA sequence specific for human RII{alpha}. Alternatively, they were incubated with a control siRNA specific for the rodent form of this protein. (A) Cells were fixed and processed for immunofluorescence with antibodies specific for RII{alpha} and C{alpha}. Cells incubated with siRNA specific for human RII{alpha} were outlined in the image corresponding to C{alpha} immunostaining. Bars, 10 µm. (B) Cells were lysed and processed for immunoblotting to detect both RII{alpha} and C{alpha}. ß'-COP was detected to normalize the amount of protein loaded. (C) Cells were homogenized and total microsomal membranes were prepared. Membranes were lysed and processed for immunoblotting to detect C{alpha}. In this case, protein disulfide isomerase (PDI) was used for normalization.

 

Figure 3
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  		Fig. 3. Golgi disassembly induced by RII{alpha} depletion. HeLa cells stably expressing galactosyltransferase-CFP (GalTfase) were incubated for 80 hours with siRNA designed to inhibit the synthesis of either the human or the rodent form of RII{alpha}. (A) Cells were fixed and processed for indirect immunofluorescence with antibodies against the indicated Golgi proteins. Bars, 10 µm. (B) Golgi-associated fluorescence and the average number of Golgi fragments per cell was determined from cells (n=12) expressing GalTfase-CFP and incubated or not (-) with either a control siRNA against the rodent form of RII{alpha} or, alternatively, an siRNA against the human version of this protein.

 

Figure 4
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  		Fig. 4. Ultrastructural changes in Golgi organization during RII{alpha} depletion. HeLa cells were plated on a dish containing a single coverslip and incubated for 80 hours with siRNA specific for human RII{alpha}. The efficiency of depletion in the culture was first evaluated by immunofluorescence detection of RII{alpha} in cells attached to the coverslip. The rest of the cells were then fixed and processed for transmission electron microscopy. (A) General view of the cytoplasm of RII{alpha}-depleted cells. (B-D) Different Golgi stacks are shown. They appear as whorls composed of multiple concentrically arrayed cisternae. Cisternae appear dilated and fragmented. Bars, 0.5 µm.

 

Figure 5
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  		Fig. 5. Assessment of Golgi polarization after disassembly. Non-transfected HeLa cells were incubated (+) or not (-) for 80 hours with siRNA designed to inhibit the synthesis of RII{alpha}. They were fixed and processed for indirect immunofluorescence with antibodies against the indicated endogenous Golgi proteins: giantin to label the cis-Golgi compartment and the TGN marker TGN46. Areas of colocalization (<10% total pixels) are white in the colocalization image. Bar, 10 µm.

 

Figure 6
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  		Fig. 6. Golgi disruption caused by PKA displacement. (A) Cells stably expressing galactosyltransferase-CFP (GalTfase) were transfected with cDNA encoding either a wild-type Ht31-GFP construct or the mutated form Ht31P-GFP. 72 hours post transfection, they were fixed and processed for immunofluorescence with anti-RII{alpha} antibody. Bar, 10 µm. (B) Cells expressing the indicated Ht31-GFP construct were fixed and processed for immunofluorescence with anti-giantin antibody. Golgi-associated fluorescence and the average number (± s.e.m.) of Golgi fragments per cell were determined (n=12).

 

Figure 7
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  		Fig. 7. Golgi disruption caused by treatment with PKA inhibitors. (A) Non-transfected cells were incubated or not (-) with 20 µM of either H89 or myristoyl-PKI (mPKI) for 2 hours at 37°C. They were fixed and processed for immunofluorescence with anti-giantin antibody. (B) The average number (± s.e.m.) of Golgi fragments per cell (n=25) was determined from non-transfected cells incubated for 2 hours at 37°C with the indicated concentrations of each inhibitor and stained for immunofluorescence with anti-giantin antibody. (C) Golgi-associated fluorescence was determined from cells (n=20) incubated or not (-) for 2 hours at 37°C with 20 µM of each inhibitor. (D) Cells stably expressing GalTfase-CFP were similarly incubated, fixed and processed for immunofluorescence with anti-giantin antibody. Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Ultrastructural changes in Golgi organization following PKA inhibition. Cells were incubated at 37°C with 20 µM of either H89 (A-D) or myristoyl-PKI (E) for 2 hours before fixation and processing for transmission electron microscopy. (F) The Golgi ribbon of a control, untreated cell is shown for comparison. Bars, 0.5 µm.

 

Figure 9
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  		Fig. 9. Role of PKA in the organization of ER exit sites. (A) Presence of PKA at the ER exit sites. Cells overexpressing CFP-tagged forms of the indicated mutated versions of Sar1p were fixed and processed for immunofluorescence with antibodies to C{alpha}. (B) PKA displacement does not affect the distribution of ER exit sites. Cells incubated for 80 hours with siRNA to target either the human or the rodent form of RII{alpha} were fixed and processed for immunofluorescence with antibodies to Sec13p. Staining of the centrosome is indicated with arrowheads. Bars, 10 µm.

 

Figure 10
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  		Fig. 10. PKA recruitment during Golgi reconstruction. Cells stably expressing GalTfase-CFP were incubated with 2 µg/ml BFA for 1 hour at 37°C and then allowed to recover for the indicated time periods. They were fixed and processed for immunofluorescence with anti-RII{alpha} antibody. Newly formed Golgi fragments containing (arrowheads) or not (arrows) associated RII{alpha} are indicated. Bar, 10 µm.

 

Figure 11
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  		Fig. 11. Golgi reconstruction abrogated by PKA inhibition. (A) Cells stably expressing GalTfase-CFP were incubated with 2 µg/ml BFA for 1 hour at 37°C. During the last 20 minutes of BFA treatment 20 µM H89 was added to the medium. Incubation continued in the presence of H89 but in the absence of BFA for the indicated time periods. Additionally, after 3 hours of BFA reversal in the presence of H89 cells were incubated in medium with no inhibitor for 1 hour (3 h + 1 h). Bars, 10 µm. (B) Non-transfected cells were incubated with 2 µg/ml BFA for 1 hour at 37°C before fixation and processing for electron microscopy. A vesicular-tubular cluster believed to represent the starting structure for Golgi reassembly (Bannykh et al., 2005Go) is shown. (C) Cells were allowed to recover from BFA treatment for 3 hours at 37°C in the continuous presence of 20 µM H89 before fixation and processing. In this case, whorl-like structures accumulate in the perinuclear region. Bars, 0.5 µm.

 

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