Article |
Address correspondence to W. James Nelson, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305-5345. Tel.: (650) 725-7596. Fax: (650) 498-5286. E-mail: wjnelson{at}stanford.edu
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Abstract |
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Key Words: cell polarity; Golgi apparatus/secretion; cell membrane/metabolism; intercellular junctions/physiology; intracellular membranes/metabolism
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Introduction |
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Molecular mechanisms that establish polarized membrane growth domains are being identified. Exocytosis involves SNARE-mediated proteinprotein interactions between transport vesicles and plasma membranes (for review see Jahn and Südhof, 1999). In polarized epithelial cells, evidence that t-SNAREs are localized to different plasma membrane domains has been presented (Gaisano et al., 1996; Low et al., 1996; Delgrossi et al., 1997; Fujita et al., 1998), and it is likely that different SNAREs are involved in exocytosis to apical and basal-lateral membrane domains (Ikonen et al., 1995; Low et al., 1998). However, during directed membrane growth in budding yeast and developing neurons there is no spatial correlation between the distribution of t-SNAREs and sites of exocytosis (Brennwald et al., 1994; Garcia et al., 1995). Therefore, it seems that t-SNAREs are necessary but not sufficient for formation of membrane growth domains.
The first component of the exocytic machinery identified at sites of polarized membrane insertion was the Sec6/8 complex (or "exocyst," in yeast) (TerBush and Novick, 1995), an 750-kD complex consisting of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) (Hsu et al., 1996; TerBush et al., 1996). The Sec6/8 complex is spatially associated with polarized membrane growth areas in budding yeast (TerBush and Novick, 1995), outgrowth areas in neurons (Hazuka et al., 1999), and growing lateral membranes in epithelial cells (Grindstaff et al., 1998). In MDCK cells, introduction of function-blocking Sec8 antibodies into permeabilized cells blocked targeted delivery of a lateral, but not an apical membrane protein (Grindstaff et al., 1998). Furthermore, overexpression of mutant Sec10 protein lacking its COOH terminus inhibited NGF-induced membrane outgrowth in PC12 cells (Vega and Hsu, 2001). In budding yeast, mutations that result in disorganization of the exocyst complex inhibit polarized membrane insertion (Novick et al., 1981). Thus, as in budding yeast, the mammalian Sec6/8 complex is likely to be an essential spatial determinant for polarized membrane growth.
To examine how Sec6/8 complex distribution and function in membrane growth are regulated, we compared two cell types that differ in their organization of intercellular junctions, and by extension, their requirement for polarized membrane growth. We report that Sec6/8 complex is present on both TGN and plasma membranes in nonpolarized fibroblasts and epithelioid cells, but that the relative proportion on these membranes is dependent on both active exocytosis and the complexity of intercellular junctions. We report that Sec6/8 is involved in several steps in exocytosis between TGN and plasma membrane.
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Results |
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Sec6/8 complex is primarily associated with the TGN in NRK-49F cells
To identify all membrane-associated pools of Sec6/8 complex in fibroblasts, cells were fractionated by isopycnic centrifugation through linear iodixanol gradients. Sec6, Sec8, and Exo70 from NRK-49F cells were recovered in four distinct peaks, with buoyant densities of 1.04, 1.10, 1.14, and 1.20 g/ml, respectively (Fig. 3 A). A fourth subunit of Sec6/8 complex, Sec5, was also recovered in the same four peaks (unpublished data). The Sec6/8 complex peak recovered at 1.20 g/ml constitutes a relatively minor fraction of the total Sec6/8 complex (
1020%). Since this peak does not cofractionate with any membrane protein markers, we conclude that it most likely represents a cytosolic pool.
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Fractionation of NRK-52E cells in iodixanol gradients revealed a relatively simple distribution of Sec6/8 complex compared with that in NRK-49F cells (Fig. 3 B). The majority of Sec6/8 complex (7075%) was recovered in a single peak with a density of 1.04 g/ml. Since this peak cofractionates with the most abundant pool of E-cadherin (45%) and has the same density as that of plasma membrane from NRK-49F cells, we conclude that most of Sec6/8 complex in NRK-52E cells is bound to plasma membrane. In contrast to NRK-49F cells, only a relatively minor amount of Sec6/8 complex (
2025%) was recovered in fractions in the density range 1.081.16 g/ml that contain the majority of VAMP4.
There is significant disparity between the spatial distribution of Sec6/8 complex (Fig. 2 A) and cell fractionation data demonstrating association of Sec6/8 complex with TGN/endosome membranes in NRK-49F cells (Fig. 3 A). It is possible that Sec6/8 complex epitopes are exposed at plasma membrane cellcell contacts, but masked when the Sec6/8 complex is bound to TGN/endosomes. To address this possibility, we analyzed a panel of monoclonal antibodies to Sec6 and Sec8. This screen revealed antibodies (mAbs 2E9, 8F9, and 8F12) that stained Sec6/8 complex at plasma membrane cellcell contact sites, and other antibodies (mAbs 3F3, 9E9, 9H5, 10C3, and 15C2) that stained Sec6/8 complex in a perinuclear compartment (Fig. 4 A). These staining patterns were mutually exclusive. To verify that the observed staining was specific for Sec6 and Sec8, each monoclonal antibody was tested by immunoblotting detergent extracts of NRK cells (Fig. 4 B).
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Additional evidence that Sec6/8 complex is primarily associated with TGN and not endosomes was obtained by determining whether Sec6-positive compartments were accessible to endocytic (Texas red transferrin; Fig. 6 A) or exocytic (green fluorescent protein [GFP]vesicular stomatitis virus G protein [VSVG] protein; Fig. 6 B) cargo. Internalization of Texas red transferrin for 5 min labeled a population of vesicular structures in the cell periphery that were negative for Sec6 (Fig. 6 A). Following a 30 min (or 60 min; unpublished data) uptake, transferrin labeled a perinuclear tubulovesicular compartment that was in the same general vicinity as membranes containing Sec6/8 complex. However, there was no overlap in the distributions of Sec6 and transferrin.
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The relationship between TGN and plasma membrane Sec6/8 complexes
At steady-state, membrane-bound Sec6/8 complexes are associated with both TGN (6070%) and plasma membrane cellcell contact sites (2030%) in NRK-49F cells, which raises the question of whether a relationship exists between these different pools.
To investigate a possible precursor product relationship between TGN- and plasma membranebound Sec6/8 complexes, we relied on our ability to resolve these organelles by isopycnic centrifugation in linear iodixanol gradients (Fig. 3). NRK-49F cells were pulse-labeled with 35S-methionine/cysteine, chased for times up to 2 h, homogenized, and fractionated. After detergent lysis of gradient fractions, Sec8 and P-cadherin were sequentially immunoprecipitated from each fraction. Immediately after metabolic radiolabeling, >90% of labeled P-cadherin is recovered in fractions 1322, and thus cofractionates with marker proteins of the ER (BiP) and TGN (VAMP4/furin) (see Fig. 3 A). Less than 10% of newly synthesized P-cadherin is recovered in low density fractions containing plasma membrane markers such as syntaxin4. After 1 h chase, 30% of labeled P-cadherin had reached plasma membrane fractions, and by 2 h chase nearly 70% was found in plasma membrane fractions (Fig. 7 A). Time-dependent increases in plasma membrane P-cadherin were balanced by concomitant decreases of P-cadherin in ER/TGN fractions and coincide with established kinetics for movement of cargo from the ER to the plasma membrane in NRK cells (Green et al., 1987).
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To further investigate a possible relationship between TGN- and plasma membraneassociated Sec6/8 complexes, the fate of newly synthesized Sec8 was determined in cells in which vesicle trafficking from the TGN was inhibited by brefeldin A (BFA). Brief exposure of NRK cells to BFA resulted in loss of TGN membrane staining of Sec6 (Fig. 8 A). This loss of staining does not reflect dissociation of Sec6/8 complex from TGN membranes, as iodixanol gradient fractionation of membranes from BFA-treated cells still shows cofractionation of Sec6/8 complex with TGN markers (Fig. 8 B). Note that BFA treatment only slightly reduced the amount of TGN-associated Sec6/8 complex, but had no effect on the association of Sec6/8 complex with plasma membrane (compare Figs. 3 A and 8 B). We conclude that BFA treatment does not significantly reduce steady-state association of Sec6/8 complex with either the TGN or plasma membrane cellcell contacts, but affects TGN structure to render Sec6 epitopes inaccessible for immunofluorescence microscopy.
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PKD/PKCµ was recently shown to be present in exocytic compartments of the TGN and expression of a kinase-inactive mutant (PKD-K618N) caused tubulation of the Golgi complex and arrested post-Golgi trafficking of VSVG in HeLa cells (Liljedahl et al., 2001). We examined effects of PKD-K618N expression on Sec6/8 complex distribution in NRK-52E cells and MDCK cells. Expression of PKD-K618N caused extensive tubulation in HeLa cells (unpublished data), similar to results reported previously (Liljedahl et al., 2001). When GFP-PKD-K618N was transiently expressed in NRK-52E cells, the mutant kinase colocalized with Sec6 in a perinuclear region, strengthening our conclusion that Sec6/8 complex is associated with a TGN compartment involved in exocytic protein trafficking (Fig. 9). A small amount of GFP-PKD-K618N was also present in peripheral vesicular structures that did not contain Sec6. Importantly, expression of GFP-PKD-K618N in these cells caused an expansion of the TGN and led to a significant increase in the level of Sec6 associated with this organelle compared with nontransfected neighboring cells (Fig. 9). Significantly, transient overexpression of GFP-PKD-K618N, combined with incubation of cells at 19°C for 2 h, resulted in accumulation of a pool of Sec6 in the TGN of MDCK cells (Fig. 9). Note that the low temperature incubation augmented TGN accumulation of Sec6 compared with transfected cells incubated at 37°C (unpublished data), but low temperature incubation alone was not sufficient to cause Sec6 accumulation on the TGN (Fig. 9, nontransfected cells). We conclude that although incubation of cells at 19°C slows exocytic trafficking in MDCK cells, association of Sec6/8 complex with the TGN is dependent on a process regulated by PKD, and that inactivation of this kinase is required for stable TGN binding of Sec6/8 complex in these cells.
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Addition of IgG from a nonimmunized mouse to semiintact cells had no inhibitory effect on delivery of ts-G-GFP to plasma membrane (Fig. 10 A). Approximately 58% of ts-G-GFP was present at the cell periphery, where it colocalized with a plasma membrane marker (ZO-1) (Fig. 10 D). Only 20% remained in the perinuclear region and the balance (
22%) was in the cytoplasm. Therefore, the apparent efficiency of TGN-to-plasma membrane delivery of ts-G-GFP in digitonin-permeabilized NRK-52E cells was similar to that in intact cells.
Antibodies directed against Sec6/8 epitopes exposed at the plasma membrane ("PM IgG") were tested as inhibitors of ts-G-GFP delivery to the plasma membrane (Fig. 10, B and D). Note that one of the antibodies in this group (mAb 2E9) was previously shown by us to inhibit delivery of basal-lateral low density lipoprotein receptors to the plasma membrane in MDCK cells (Grindstaff et al., 1998), so we anticipated an inhibitory effect on transport of VSV G protein (also a basal-laterally sorted protein). Quantitation of ts-G-GFP distribution in permeabilized NRK cells incubated with PM IgG shows that 20% remains in the perinuclear region,
35% is in the cytoplasm, and
45% is at the cell periphery (Fig. 10 B). Although a significant amount of ts-G-GFP appeared to be trapped between TGN and plasma membrane, much of the cargo was delivered to peripheral region. However, visual inspection of peripheral regions of cells incubated with control versus PM IgG reveals differences in distribution of ts-G-GFP (Fig. 10 D). Peripheral ts-G-GFP in control cells almost always (13/15 cells examined) has a continuous, linear distribution and colocalizes with plasma membrane ZO-1 staining. In contrast, peripheral ts-G-GFP in cells incubated with PM IgG frequently (9/18 cells examined) appears discontinuous or punctate (Fig. 10 D). In other cells, peripheral ts-G-GFP has a continuous distribution that appears to be just beneath plasma membrane ZO-1 staining, distinct from control cells in which cargo and ZO-1 distributions overlap (Fig. 10 D).
When TGN Sec6 IgG cocktail was included in the transport assay, only 22% of ts-G-GFP arrived in the cell periphery and cargo was primarily (50%) retained in the perinuclear region (Fig. 10 C). The balance of ts-G-GFP (
28%) was in cytoplasmic regions (Fig. 10 C). This latter amount is similar to control levels and indicates that TGN IgG cocktail acts to inhibit release of cargo-laden transport vesicles from the perinuclear vicinity, rather than trafficking of these vesicles to the cell periphery. The fact that some ts-G-GFP (
12%) arrives at the cell periphery indicates that a fraction of cargo was insensitive to function-blocking antibodies or had already been packaged for post-TGN delivery before antibody addition.
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Discussion |
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Regulation of Sec6/8 localization to TGN and plasma membrane
Previous studies indicated that the Sec6/8 complex functions at the plasma membrane (TerBush and Novick, 1995; Grindstaff et al., 1998), perhaps to tether transport vesicles during exocytosis. So our finding that a major fraction of the complex associates with the TGN in NRK-49F cells was surprising. A relationship between Sec6/8 complex pools on TGN and plasma membrane was investigated by a simple metabolic pulse-chase approach. In the presence of BFA, newly synthesized Sec6/8 complex appeared on TGN, but not plasma membrane, indicating that active exocytosis is required for recruitment of Sec6/8 complex to plasma membrane. In the absence of BFA, steady-state distribution of Sec6/8 complex between TGN and plasma membrane was reached within 30 min. This time is sufficient for arrival of some TGN-derived vesicles at the plasma membrane, but probably insufficient for steady-state to be achieved if only a TGN-derived pool of Sec6/8 complex contributed to the plasma membranebound pool. It is more likely that plasma membrane Sec6/8 complex is also derived from a cytosolic pool. At present, it is not known how Sec6/8 complex binds membranes. However, since Sec6/8 complex remains TGN-bound in the presence of BFA we conclude that this interaction is not controlled directly by the Sec7 family of guanine nucleotide exchange factors that are the targets of BFA action (Jackson and Casanova, 2000).
We note that two other studies were published recently that reported localization of Sec6/8 complex to a perinuclear compartment in pancreatic acinar cells (Shin et al., 2000) and PC12 cells (Vega and Hsu, 2001). However, a detailed analysis of membrane binding of Sec6/8 complex or colocalization with membrane markers was not rigorously pursued. Our finding that a pool of Sec6/8 complex associates with TGN membranes in NRK-49F cells is different from data on MDCK cells reported previously by us (Grindstaff et al., 1998). Immunofluorescent staining of polarized MDCK cells showed Sec6/8 complex almost entirely associated with plasma membrane in close proximity to tight junctions. Expression of a kinase-inactive mutant of PKD (PKD-K618N), which efficiently inhibits protein exit from TGN (Liljedahl et al., 2001), caused Sec6/8 complex to accumulate in the TGN of MDCK cells; this accumulation was augmented in PKD-K618Nexpressing cells following incubation at 19°C. These data show that exocytosis in MDCK cells, as in NRK cells, is required to develop the steady-state distribution of Sec6/8 complex.
Sec6/8 functions in the exocytic pathway at the TGN and plasma membrane
A function for plasma membrane-bound Sec6/8 complex in exocytic vesicle delivery has been demonstrated genetically in yeast (Novick et al., 1981; Finger et al., 1998; Guo et al., 1999) and biochemically in mammalian cells (Grindstaff et al., 1998), but a role for Sec6/8 complex in TGN was unclear in those studies. We investigated the role of TGN-bound Sec6/8 complex in exocytosis using an antibody-blocking assay similar to that used by us previously for plasma membranebound Sec6/8 complex, combined with a morphological analysis of the distribution of cargo (ts-G-GFP) in late stages of the exocytic pathway. Addition of antibodies specific for the TGN Sec6/8 complex blocked ts-G-GFP transport from the perinuclear region to the cell periphery; importantly, a nonspecific antibody and antibodies specific for the plasma membrane Sec6/8 complex did not affect cargo exit from the TGN. We speculate that antibodies bound the Sec6/8 complex and sterically inhibited protein interactions normally required for cargo exocytosis, although the precise function of Sec6/8 complex on the TGN is unknown at present. It is possible that vesicles continue to bud, but cluster near the TGN because bound antibodies prevent translocation to the cell periphery. We note that cargo protein (ts-G-GFP) transits through TGN compartment that contains Sec6/8 complex, and that PKD and Sec6/8, both of which are required for cargo exit from TGN, colocalize in this TGN subcompartment. Therefore, it is tempting to speculate that this subcompartment represents a sorting domain for a class of exocytic membrane proteins. In this context, Fölsch et al. (2001) have also concluded that µ1B, which is required for sorting of the same cargo, is localized in the TGN in a subcompartment different from that of µ1A and furin.
When ts-G-GFP was released from TGN in the presence of antibodies to the plasma membrane Sec6/8 complex it accumulated in aggregates that were frequently localized adjacent to plasma membrane, but did not costain with markers of the plasma membrane. This distribution was different from that in control cells in which ts-G-GFP had a linear distribution along the plasma membrane coincident with that of ZO-1. We suggest that antibodies to the plasma membrane Sec6/8 complex sterically inhibited protein interactions required for docking of post-TGN exocytic vesicles with the plasma membrane, and as a consequence vesicles containing ts-G-GFP stacked up at these sites. This result provides a visual affirmation of our previous results in which antibodies to the plasma membrane Sec6/8 complex blocked basal-lateral plasma membrane delivery of low density lipoprotein receptor in polarized MDCK cells (Grindstaff et al., 1998). It is noteworthy that in MDCK cells VSVG protein is also delivered to the basal-lateral membrane, indicating that the Sec6/8 complex is required for delivery of vesicles containing this class of proteins in both polarized and nonpolarized cells. However, analysis of additional apically and basal-laterally targeted membrane proteins will be required to show this unequivocally.
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Materials and methods |
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Cell culture methodology
NRK-49F (ATCC CRL 1570) and NRK-52E (ATCC CRL 1571) cells were maintained in DME supplemented with 10% fetal bovine serum. In some experiments, cells were incubated for 530 min in 35 µg/ml BFA (Calbiochem), or medium containing 20 µg/ml nocodazole (Sigma-Aldrich) for 2 h (Reaves and Banting, 1992). For internalization of Texas red transferrin, cells were preincubated at 37°C in serum-free DME containing 3% BSA for 60 min, then incubated at 0°C for 60 min in the same medium supplemented with 100 µg/ml Texas red transferrin. After three washes with medium, internalization of fluorescent transferrin was initiated by addition of warm medium and incubation of cells at 37°C for different lengths of time. Transfection of NRK-49F and NRK-52E cells was performed by electroporating cells using 10 µg DNA as described previously (Chao et al., 1999). Transfection of MDCK cells was achieved using the calcium phosphate precipitation method (Ausubel et al., 1987). Cells were fixed and processed for immunofluorescent staining 4872 h after transfection. To synchronize ts-G-GFP in different exocytic compartments, transfected cells were incubated at 40°C for 12 h to accumulate ts-G-GFP in the ER, shifted to 32°C for 7.5 min to allow ts-G-GFP to fold, and subsequently transferred to 19°C for 2 h to accumulate protein in the TGN. Trafficking of protein from the TGN to plasma membrane was initiated by shifting cultures from 19°C to 32°C.
Cell fractionation in iodixanol gradients
Cell homogenization and separation of membrane and cytosolic fractions was achieved by centrifugation in 30% (wt/vol) iodixanol (Grindstaff et al., 1998). Separation of different membrane compartments was achieved by centrifugation in three-step 102030% (wt/vol) iodixanol gradients. Briefly, one-third of the postnuclear supernatant was mixed with Opti-Prep (60% (wt/vol) iodixanol (Nycomed) and homogenization buffer to generate solutions containing 10, 20, or 30% iodixanol. Equal volumes of these three solutions were layered in centrifuge tubes and samples were centrifuged at 353,000 g for 3 h at 4°C in a Beckman Coulter Vti65 rotor. Fractions (0.5 ml) were collected and proteins were separated by SDS-PAGE and immunoblotted (Grindstaff et al., 1998).
Immunofluorescent staining
Cells were fixed in 4% paraformaldehyde for 30 min, before or after extraction at 0°C for 10 min with 1% Triton X-100 in buffer containing 10 mM Pipes, pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.1 mg/ml RNase, 0.1 mg/ml DNase, and protease inhibitors (CSK buffer). For immunofluorescent staining of TGN/endosomes (Figs. 47, 9, and 10), cells were fixed and then permeabilized with 0.075% saponin. Monoclonal Sec6 or Sec8 antibodies (as hybridoma supernatants diluted 1:4), monoclonal syntaxin13 antibody (1:1,000), and polyclonal antibodies to E/P-cadherin (1:25), -catenin (1:500), occludin (1:500), ZO-1 (1:300), ZO-2 (1:200), VAMP4 (1:500), VAMP8 (1:100), and furin (1:100) were applied to cells for 2 h at 4°C. Fluorescein and rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) diluted at 1:200, or rhodamine-phalloidin (1:40) were applied for 1 h at 4°C. Coverslips were washed five times and mounted in VectaShield (Vector Laboratories). Samples were viewed with either a ZEISS Axioplan microscope (100x objective) or a Molecular Dynamics MultiProbe 2010 confocal laser scanning microscope (63x objective).
Metabolic pulse-chase analysis
NRK-49F cells were incubated in methionine/cysteine-free DME for 30 min in the absence or presence of 5 µg/ml BFA. Cells were metabolically labeled with 200 µCi/ml 35S-proMix (Amersham Pharmacia Biotech) for 30 min, then chased for 0, 1, 2, or 3 h in DME containing 0.2 mM methionine/cysteine. To determine the overall stability of Sec8 in NRK-49F cells, cells were extracted in CSK for 30 min at 4°C. To determine the relative amount of pulse-labeled Sec8 and P-cadherin associated with different membrane compartments during the chase, cells were homogenized at each time point and postnuclear supernatants were fractionated in linear three-step (102030%) iodixanol gradients. Fractions (0.5 ml) were collected and processed for immunoprecipitation with Sec8 antibodies (5C3, 2E12, and 10C2) or a pan-cadherin rabbit polyclonal antibody (E2) (Pasdar and Nelson, 1989).
Morphological assay for plasma membrane delivery of ts-G-GFP
NRK-52E cells were transfected with plasmid encoding ts-G-GFP and protein was accumulated in the TGN as described above. Cells were permeabilized with digitonin (30 µg/ml) and incubated in transport mix containing control or anti-Sec6/8 antibodies at 19°C for 15 min, then shifted to 32°C for 60 min. Transport mix contained 10 mg/ml bovine brain cytosol, 2.5 mM MgATP, 1.25 mM GTP, 15 mM creatine phosphate, 0.25 mg/ml creatine kinase, 1 mM DTT, and protease inhibitors in buffer containing 20 mM Hepes KOH, 90 mM KOAc, 2 mM Mg(OAc)2, 0.05 mM EGTA, and 0.9 mM CaCl2. After incubation at 32°C for 60 min, coverslips were transferred to 0°C and processed for immunofluorescence as above. Cells were incubated with antibodies to furin (to stain TGN), ZO-1 (to stain plasma membrane), or with secondary antimouse antibodies (to stain control and AntiSec6/8 antibodies introduced into permeabilized cells). Serial confocal sections were collected and analyzed using ImageSpace, v. 3.2 software (Molecular Dynamics). Cell periphery, perinuclear regions, and the region in between ("cytoplasm") were manually outlined and fluorescent signals in these areas were quantified as described previously (Pepperkok et al., 1993; Hirschberg et al., 1998).
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Footnotes |
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Kent Grindstaff's present address is Xenoport, Inc., Department of Discovery Biology, 2631 Hanover St., Palo Alto, CA 94304.
Jessica Wright's present address is Department of Biological Sciences, Stanford University, Stanford, CA 94305.
* Abbreviations used in this paper: BFA, brefeldin A; GFP, green fluorescent protein; NRK, normal rat kidney; ts, temperature sensitive; VSVG, vesicular stomatitis virus G protein.
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Acknowledgments |
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Submitted: 23 July 2001
Revised: 28 September 2001
Accepted: 1 October 2001
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References |
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