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Address correspondence to Ira Mellman, Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, 333 Cedar St., PO Box 208002, New Haven, CT 06520-8002. Tel.: (203) 785-4303. Fax: (203) 785-4301. email: ira.mellman{at}yale.edu
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Abstract |
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Key Words: Rab8; AP-1; sorting; polarity; MDCK
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Introduction |
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Some of the components involved in basolateral transport have recently been identified. Among these is an epithelial cellspecific form of the AP-1 clathrin adaptor complex, AP-1B (Fölsch et al., 1999, 2003; Ohno et al., 1999). This tetrameric complex is closely related to the ubiquitously expressed AP-1A complex. The two differ only by substitution of the ubiquitous 50-kD µ1A subunit (AP-1A) with a homologous µ1B subunit (AP-1B), whose expression is limited to epithelia. The presence of µ1B confers the ability to recognize and mediate basolateral transport of membrane proteins bearing tyrosine-dependent targeting signals (e.g., LDLR, vesicular stomatitis virus G-protein [VSV-G], and asialoglycoprotein receptor) and at least one tyrosine-independent signal (transferrin [Tfn] receptor; Fölsch et al., 1999; Sugimoto et al., 2002). Interestingly, AP-1B does not program the basolateral targeting of proteins bearing dileucine-type signals (e.g., FcRII-B2), as these reach the basolateral surface of µ1B-negative LLC-PK1 cells (Roush et al., 1998; Fölsch et al., 1999). The precise site of AP-1B action is unknown, but it may act to control polarized sorting on both the endocytic and biosynthetic pathways, i.e., in endosomes and the TGN (Gan et al., 2002).
Clearly, other components must play a role in the formation and delivery of basolateral transport carriers. The Rho family GTPase Cdc42 has been found to play an essential role in basolateral targeting in MDCK cells (Kroschewski et al., 1999; Cohen et al., 2001; Musch et al., 2001), although it is unclear if this role is limited to AP-1Bdependent or independent pathways. Similarly, the multi-subunit exocyst complex, first identified in yeast as being required for tethering secretory vesicles at the bud tip (TerBush et al., 1996), has also been implicated in basolateral transport. In MDCK cells, antibodies to two mammalian exocyst subunits (Sec6 and Sec8) partially inhibited the insertion of LDLR in the basolateral plasma membrane (Grindstaff et al., 1998; Yeaman et al., 2001; Moskalenko et al., 2002). Because LDLR has been identified as AP-1Bdependent cargo (Fölsch et al., 1999), it seems possible that the exocyst is involved in the AP-1B pathway.
The small GTPases that regulate exocyst function might therefore have a role in AP-1Bdependent sorting by supervising the organization of components required for sorting or vesicle delivery. In yeast, the Rab family member Sec4p is localized to transport vesicles and interacts genetically with the exocyst to facilitate the targeting of secretory vesicles to the plasma membrane (Guo et al., 1997, 1999). Rab8 is among the closest mammalian homologues to Sec4p and has, in fact, previously been associated with transport from the TGN to the plasma membrane in neurons and MDCK cells (Huber et al., 1993, 1995; Moritz et al., 2001). A role in basolateral transport was suggested by Rab8's association with basolateral vesicles upon cell fractionation, although the functional significance of this observation has remained unclear; only a slight inhibition of transport was seen in permeabilized MDCK cells using a peptide from the Rab8 hypervariable COOH-terminal domain (Huber et al., 1993).
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Results |
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As shown in Fig. 1 A, expression of the constitutively active allele of Rab8 (Rab8Q67L) caused a great majority of VSV-GGFP in any one cell to be expressed apically, in contrast to the lateral or basolateral expression obtained in cells injected with the dominant-negative Rab8 cDNA (Rab8T22N) or with the VSV-GGFP cDNA alone. Overexpression of wild-type Rab8 elicited a phenotype similar to Rab8Q67L (unpublished data). In both cases, the effects were apparently selective for newly synthesized proteins and did not reflect an overall reorganization because the localization of the endogenous basolateral marker gp58 remained unchanged. Moreover, the expected apical localization of VSV-GG3 variant was not affected (Fig. 1 B), suggesting that Rab8 activation selectively affected basolateral transport.
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Rab8Q67L expression may have caused ectopic expression of VSV-G, but it also might have had an effect on the overall efficiency of VSV-G transport, a feature that might have been missed by fluorescence microscopy alone. Therefore, we performed FACS® analysis on nonpermeabilized cells to compare the efficiency of VSV-G surface arrival in the presence or absence of activated Rab8, using the TK-G antibody. To ensure that a substantial population of cells expressed VSV-G with or without Rab8, MDCK cells were infected with recombinant adenoviruses encoding the VSV-GGFP and Rab8Q67L genes described above. The FACS® analysis revealed that the level of cell surface VSV-G expression was unchanged by activated Rab8 expression (Fig. 1 D), indicating that Rab8Q67L does not inhibit the delivery of VSV-G to the plasma membrane. Although only 50% of the cells in the population expressed both markers, we noted no decrease in the level of TK-G binding. Together, these data suggested that Rab8 has a selective role in the sorting or targeting of basolaterally directed cargo, and that expression of Rab8Q67L does not cause overall defects in polarity or the secretory pathway.
Newly synthesized VSV-G is directly missorted to the apical surface in cells expressing activated Rab8
We sought to determine if activated Rab8 acted to directly missort newly synthesized VSV-G on the secretory pathway or indirectly after initial basolateral insertion and missorting during endocytosis and recycling. For this purpose, filter-grown MDCK cells were doubly infected with adenoviruses encoding ts045 VSV-GGFP or activated Rab8, and the surface appearance of VSV-G was analyzed by pulse-chase radiolabeling and surface biotinylation. As expected, in the absence of exogenous Rab8 expression, VSV-G appeared at the basolateral surface, reaching a plateau within 6090 min and decreasing (in some experiments) thereafter (Fig. 2). Importantly, little VSV-G was detected at the apical surface at early or late times of chase. However, in cells coinfected with activated Rab8, VSV-G appeared simultaneously at both the apical and basolateral surfaces at times as early as 30 min of chase. The delivery of VSV-G to the apical surface without prior appearance at the basolateral surface strongly suggested that missorting occurred on the secretory pathway upon exit from the TGN rather than in endosomes after endocytosis from the basolateral domain.
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Expression of mutant Rab8 missorts only AP-1B cargo
To determine whether Rab8 caused the missorting of all basolateral proteins regardless of their specific targeting determinants, we analyzed basolateral reporters that have been shown to be AP-1Bdependent or independent using the microinjection assay. The immunoglobulin Fc receptor (FcR) relies on a dileucine motif for localization (Matter et al., 1994) and was found to reach the basolateral surface of polarized pig kidney epithelial (LLC-PK1) cells even in the absence of µ1B expression (Roush et al., 1998; Fölsch et al., 1999). LDLR, on other hand, depends on tyrosine motifs for basolateral sorting (Matter et al., 1992) that physically interact with AP-1B adaptors and require µ1B expression for basolateral targeting in LLC-PK1 cells (Ohno et al., 1998; Fölsch et al., 1999, 2001; Sugimoto et al., 2002). For these experiments, a Rab8-GFP was used to monitor expression while surface polarity of FcR and LDLR was detected by staining nonpermeabilized cells with antibodies to the ectodomain of each receptor.
It was necessary to alter the temperature shift protocol used earlier in order to accommodate reporter proteins that, unlike ts045 VSV-G, could not be accumulated in the ER at 40°C before assay. Thus, after microinjection, cells were maintained at 20°C for 2.5 h to accumulate each reporter (i.e., FcR and LDLR) in the TGN before release by shifting the temperature up to 31°C. As a control, we first established that this protocol resulted still in the missorting of ts045 VSV-G in mutant Rab8-expressing cells (unpublished data). After microinjection of wild-type or mutant Rab8-GFP together with LDLR or FcR cDNAs, the receptors were accumulated in the Golgi complex by incubation at 20°C (2.5 h) followed by release at 37°C in the presence of cycloheximide. As found for VSV-G, which also uses an AP-1Bdependent tyrosine-based sorting motif (Thomas and Roth, 1994; Fölsch et al., 2003), LDLR was missorted to the apical surface in the presence of activated Rab8 (Fig. 3 A). In striking contrast was the behavior of FcR. As shown in Fig. 3 B, FcR remained at the basolateral surface despite the efficient expression of the active Rab8 mutant Rab8Q67L. Identical results for both receptors were obtained using non-GFP-tagged Rab8 (unpublished data). Thus, it appeared that mutant Rab8 expression affected only the polarity of AP-1Bdependent cargo.
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In a further control for the specificity of Rab8's effect, we microinjected wild-type or mutant GFP-tagged Rab11, a GTPase that has an intracellular distribution similar to that of Rab8 (Fig. 3 A) and that is also a structural relative of Rab8 and Sec4p. However, unlike Rab8Q67L, activated Rab11 (Rab11Q70L) had no effect on LDLR transport to the basolateral plasma membrane (Fig. 3 A). Thus, whatever its mechanism, the phenotype of missorting tyrosine motifcontaining basolateral proteins was specific to active Rab8, and not a general consequence of disrupting the function of Rab family proteins in the recycling endosome/TGN region of the cytoplasm.
Expression of mutant Cdc42 also missorts only AP-1B cargo
In previous work, we found that expression of mutant Cdc42, especially a Cdc42 dominant-negative allele, caused the missorting of basolateral but not apical cargo (Kroschewski et al., 1999), a finding confirmed by others (Johnson, 1999; Joberty et al., 2000; Cohen et al., 2001; Musch et al., 2001). Because this effect was observed for VSV-G, we next asked if Cdc42 might also selectively regulate the AP-1Bsorting pathway. For these experiments, we again used the 20°C temperature shift protocol to allow the use of LDLR and FcR as reporters in addition to VSV-G. As shown previously, when Cdc42 was functionally deleted by microinjection of a cDNA encoding a dominant-negative allele (Cdc42T17N), VSV-G was missorted to the apical surface of filter-grown MDCK cells (Fig. 3 C). Similar results were obtained when the polarized expression of a second AP-1B cargo protein LDLR was monitored (Fig. 3 C). However, dominant-negative Cdc42 caused no detectable alteration in the basolateral expression of FcR, an AP-1Bindependent basolateral protein (Fig. 3 C). Together, these observations strongly suggested that expression of Rab8 and dominant-negative Cdc42 selectively affected the localization of basolateral proteins that rely on AP-1B adaptor. Even though the precise mechanism by which either GTPase elicits its effects is not yet clear, it does seem that they are restricted to a common basolateral-sorting pathway.
Rab8 is localized to the perinuclear region in recycling endosomes
To understand the role of Rab8 in the AP-1Bdependent basolateral sorting pathway, we examined the localization of Rab8 in MDCK cells. Although previous work had localized Rab8 to the "Golgi region" (Huber et al., 1993; Peranen et al., 1996; Peranen and Furuhjelm, 2001), its distribution remains unclear. cDNAs encoding GFP-tagged Rab8, wild type, or Rab8Q67L were microinjected at low concentration (to provide trace labeling) into MDCK cells grown on coverslips. After fixation, the cells were colabeled with antibodies against various compartments, particularly the TGN and recycling endosomes, thought to be sites of polarized sorting. As shown in Fig. 4, wild-type Rab8 was localized to the perinuclear region, although in some cells labeling was occasionally in peripheral structures as well as possibly on plasma membrane. However, the perinuclear Rab8-GFP was excluded from structures positive for GM130, a cis-Golgi marker (Fig. 4 A), as well as for giantin, a pan-Golgi marker (unpublished data), indicating that this portion of its distribution did not reflect an association with Golgi cisternae. Similar results were obtained for Rab8Q67L-GFP (unpublished data).
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At least a fraction of Rab8 appeared to associate with recycling endosomes, evidenced by the partial colocalization of GFP-Rab8 with internalized Tfn (Sheff et al., 1999; Fig. 4 C, arrow). This coregional distribution was particularly evident in cells where the recycling endosomes were characteristically clustered in the perinuclear cytoplasm, although markers of cisternal Golgi and the TGN continued to be distinct from Rab8-GFP (unpublished data). Three-dimensional reconstruction of z-axis sections was used to further evaluate the spatial relationship of Rab8-GFP and Tfn (Fig. 4 D). Upon rotating such images and sectioning them sagittally (Fig. 4 E), it was apparent that in areas of coregionalization, the overlap between Rab8-GFP and Tfn was found throughout the volume of the structures observed (Fig. 4, D and E, yellow). Thus, the areas of overlap did not represent separate structures superimposed across different focal planes. To provide more direct evidence, immuno-EM was performed on cells prepared as in Fig. 4 D. As shown in Fig. 5 (A and B), at least a fraction of Rab8 (10-nm gold) was clearly localized to the Tfn-positive (5-nm gold) endosomes (arrows) and coated vesicle buds (arrowhead). As expected from the IF data, some Rab8 labeling was also found associated with Golgi elements. In any event, the EM data suggest that recycling endosomes may be the site of action of Rab8, and thus a possible site for sorting of AP-1Bspecific cargo.
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As shown in Fig. 7 A (top panels), expression of Rab8Q67L in AP-1A+/AP-1B- LLC-PK1 cells did not cause the dispersal of -adaptin seen in MDCK cells (which are AP-1A+/AP-1B+). Thus, it appeared that Rab8 activation had no effect on AP-1A adaptor localization or membrane recruitment.
-Adaptin staining remained similar to that of the AP-1A cargo protein TGN-38 (expressed from a cotransfected cDNA; Fig. 7 A, blue). In contrast, the localization of
-adaptin was substantially altered in LLC-PK1 cells that had been transfected with a µ1B cDNA and thus expressed functional AP-1B (Fig. 7 A, bottom, AP-1A+/AP-1B+). Notably, some
-adaptin staining did persist in the perinuclear region of AP-1Bexpressing LLC-PK1 cells. These structures most likely reflected the
-adaptin subunits of AP-1A adaptor complexes, as their staining pattern was similar to TGN-38.
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Discussion |
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Understanding the pathway controlled by AP-1B will be facilitated by identifying and characterizing the protein components with which it must collaborate. We have identified two such components, Rab8 and Cdc42. Rab8 had early on been implicated in basolateral transport in MDCK cells. This suggestion came from work demonstrating a partial inhibition of VSV-G insertion into the basolateral plasma membrane of permeabilized cells treated with a Rab8 hypervariable domain peptide (Huber et al., 1993). However, anti-Rab8 antibodies were without effect. Moreover, it remained unclear if the peptide's effect applied to all forms of basolateral transport or was selective for the AP-1B pathway (which had not yet been identified). Similarly, although Cdc42 had also been shown to be involved in basolateral transport, until now there was no indication that its effects were selective for the AP-1B pathway. The high degree of specificity exhibited by both Rab8 and Cdc42 for inhibiting AP-1Bdependent cargo strongly suggests that they functionally interact, even if indirectly, with the AP-1B complex, and that they help define a common pathway.
As a homologue of yeast Sec4p, it seems likely that Rab8 might also function together with the mammalian exocyst complex. Although we have not demonstrated such an interaction directly, it is important to note that at least two exocyst components (Sec6 and Sec8) have been associated with the delivery of AP-1B cargo (e.g., LDLR) to the basolateral surface of MDCK cells (Grindstaff et al., 1998). Moreover, expression of µ1B in LLC-PK1 cells enhances the recruitment of exocyst subunits (Sec8 and Exo70) to the TGN/recycling endosome region of AP-1Bnegative LLC-PK1 cells (Fölsch et al., 2003). Rab8, too, was found in the same region.
Our findings have one further implication for understanding polarized transport in MDCK cells. They clearly indicate that there are two distinct modes of reaching the basolateral plasma membrane. In LLC-PK1 cells (which do not express µ1B), dileucine-containing membrane proteins such as FcR are nevertheless targeted basolaterally, suggesting the existence of a second, AP-1Bindependent pathway or mechanism of sorting in these cells. The fact that in µ1B-positive MDCK cells, expression of mutant Rab8 and Cdc42 had no effect on FcR polarity suggests that a similar mechanism was simultaneously operative despite the presence of AP-1B adaptors. Although this result suggests that the AP-1Bindependent pathway reflects the formation of a distinct class of transport carriers, it remains possible that the dileucine-specific adaptor recruits its cargo to the same carriers as does AP-1B. Such a mechanism would be similar to what has recently been proposed for the selection of GGA and AP-1A cargo into clathrin-coated buds at the TGN during transport to lysosomes in mammalian cells (Hirst et al., 2000; Puertollano et al., 2001). However, it is unlikely that the AP-1Bindependent pathway in MDCK cells (or LLC-PK1 cells) reflects the mechanism of basolateral targeting in polarized cells such as hepatocytes or neurons. Such cells do not express µ1B, yet they mediate the basolateral (or somato-dendritic) delivery of membrane proteins in a fashion strictly dependent on AP-1B signals (Jareb and Banker, 1998; Koivisto et al., 2001). Similarly, in Drosophila eye disc epithelial cells, which do not express a second µ1 gene, mammalian AP-1Bdependent cargo is nevertheless targeted accurately to the basolateral surface again in a signal-dependent fashion (unpublished data).
How and where does Rab8 work? The fact that Rab8-GFP was localized to the perinuclear region of MDCK cells, as suggested previously for endogenous Rab8 (Huber et al., 1993), suggests that it exerts at least part of its function during sorting or vesicle formation. Indeed, expression of active Rab8 was found to cause missorting of newly synthesized VSV-G to the apical surface. It also caused a selective disruption of AP-1B's association with membranes in the perinuclear region; remarkably, AP-1A's association with membranes in the same region was not affected by active Rab8 expression. Because there are now clear examples (e.g., Rab9) of Rab proteins playing an accessory role in sorting or transport vesicle formation (Carroll et al., 2001), such a function for Rab8 is indeed plausible. This is not inconsistent with early findings that Rab8 can be found on immunoisolated basolateral transport vesicles (Huber et al., 1993), or that it might also work (by analogy to Sec4p) in an exocyst-mediated tethering event before fusion at the basolateral membrane.
Similar considerations apply to Cdc42. It is found in the Golgi region of MDCK cells (Erickson et al., 1996) and has also been associated with more rapid export of basolateral cargo from the perinuclear zone (Musch et al., 2001). However, Cdc42 may interact with exocyst components and also is found in a ternary complex together with Par3, Par6, and PKDµ at junctional complexes in MDCK cells (Joberty et al., 2000; Zhang et al., 2001), all suggesting a possible role in plasma membrane tethering or fusion.
Our confocal microscopy results suggest that despite the close spatial apposition of Rab8-GFP to AP-1B adaptors and recycling endosomes, the actual degree of "overlap" is limited. Based on our EM data, it would appear that Rab8 localizes extensively with Tfn-containing recycling endosomes. Indeed, Sec8 and Exo70 also appear to exhibit a similar recycling endosome-like pattern in AP-1Bexpressing cells (Fölsch et al., 2003). Such observations suggest that polarized sorting may actually occur after exit of AP-1B cargo from the TGN, perhaps in recycling endosomes or a subcompartment closely apposed to these sites. A functional relationship between the TGN and recycling endosome has long been suspected. The finding that a Rab protein controlling the transport of newly synthesized plasma membrane proteins in fact localizes to endocytic structures supports the idea that the secretory and endocytic pathways intersect (Futter et al., 1995; Harsay and Schekman, 2002). Because recycling endosomes in MDCK cells may be associated with polarized sorting during endocytosis (Hedman et al., 1987; Stoorvogel et al., 1988; Sheff et al., 1999), an intersection at this level would provide a common intracellular site at which polarity is generated. Although additional work will be required to establish this point functionally, our current experiments suggest a number of testable hypotheses.
The final issue raised concerns the mechanism of Rab8 action. The missorting phenotype was observed upon expression of a constitutively active Rab8 GTPase allele or overexpression of wild-type Rab8. No effect was observed when a dominant-negative Rab8 allele was expressed. Conceivably, the dominant-negative allele was simply inactive, as opposed to acting in a dominant-negative fashion. If true, only the active allele would be expected to interfere with the normal cycle of nucleotide binding and hydrolysis, perhaps by sequestering one or more Rab8 effectors. Such a mechanism might interfere with the proper sorting of AP-1B cargo into forming transport vesicles or might block transport vesicle formation itself. In turn, this would be expected to cause VSV-G to "leak," at least in part, into the apical pathway, as is seen for other basolateral proteins when the AP-1B pathway is not available. Alternatively, active Rab8 might somehow enhance the apical pathway itself, rendering apical missorting of proteins with basolateral targeting signals quantitatively more efficient. Finally, as is possible in the case of Cdc42, Rab8 may affect sorting or traffic via the actin cytoskeleton. Rab8 is similar to Rho family GTPases by the fact that its hypervariable domain contains only a single prenyl group (Joberty et al., 1993). Moreover, when greatly overexpressed, active Rab8 will cause the formation of dendritic extensions in neurons and even MDCK cells (although not under the conditions used here; Peranen et al., 1996). In any event, we suspect that a search for Rab8-interacting proteins, as has been accomplished for other Rab proteins, will yield important insights not only into Rab8 function in particular, but polarized sorting in general.
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Materials and methods |
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Recombinant adenovirus construction
T7-tagged Rab8 was cloned into pAdEasyTM shuttle vector at KpnIXhoI using primers 5'-GGGGTACCATGGCTAGCATGACTGGTGGACAGCAAATGGGTGCGAAGACCTACGATTACCTG-3' and 5'-CCGCTCGAGTCACAGAAGAACACATCGGAA-3'. Rab8C used primer 5'-CCCAAGCTTTCAT-CGGAAAAAGCTGCTCCTCTT-3'. Shuttle vector constructs were recombined with pAdEasyTM-1 vector as described in the Qbiogene manual, version 1.3.
Cell culture
MDCK cells were cultured in MEM (10% FBS) and plated on clear permeable Transwell polycarbonate filters (Corning Costar) at 105 cells/cm2. Cells were grown 4 d and microinjected in Hepes-buffered media after excision from filter holders. The cDNA for Rab8, LDLR, and VSV-GGFP cDNAs (0.2 mg/ml) were injected into nuclei of 400 cells using an Eppendorf Transjector microinjection system mounted on an inverted microscope (Axiovert; Carl Zeiss MicroImaging, Inc.) with a 40°C heated stage. After injection, the filters were incubated at 40°C for 2 h for ts045 VSV-G GFP expression and for retention in the ER. Cells injected with LDLR or FcR were incubated at 37°C for 1 h, at 20°C for 22.5 h, and then returned to 37°C for 2 h with 0.1 mg/ml cycloheximide. Filters were fixed in 4% PFA and processed for IF. LLC-PK1 cells were cultured in
-MEM, 10% FBS, and 1.8 mg/ml geneticin. µ1A-/µ1B+ fibroblasts (Eskelinen et al., 2002) were grown in Dulbecco's minimum essential medium (DMEM), 10% FBS, and 200 µg/ml hygromycin. For IF, cells were seeded on Alcian bluecoated coverslips and cultured for 3 d before transfection. MDCK-Tfn receptor (MDCKT) stable cells were cultured in DMEM, 10% FBS, and 0.5 g/ml geneticin as described previously (Sheff et al., 1999.)
Recombinant adenoviruses and transfection
The ts045 VSV-GGFP and apical variant were gifts from Patrick Keller (European Molecular Biology Laboratory, Heidelberg, Germany; Keller et al., 2001). Cells were infected 24 h before analysis by IF or pulse-chase biotinylation. Cells were infected at 4 plaque-forming units/cell. Propagation and generation of recombinant adenoviruses were performed as described in the pAdEasyTM vector protocol (Qbiogene). LipofectAMINETM (Invitrogen) was used for transient transfection in LLC-PK1 and embryonic fibroblast cells.
IF microscopy
For total cell staining, cells were fixed in 4% PFA for 15 min followed by 510 min of blocking/permeabilization in PBS with 10% goat serum (GS) and 0.25% saponin. Cells were incubated for 1 h with primary antibody diluted in blocking solution (BS). The cells were washed three times for 10 min and incubated for 30 min in secondary antibody (with appropriate Alexa® Fluors; (Molecular Probes, Inc.). The cells were washed three times for 10 min in PBS and mounted in Mowiol/DABCO/glycerol solution. For surface staining, after fixation, cells were blocked in 10% GS before labeling with the first primary antibody for 1 h. Cells were then washed three times for 10 min in BS. The cells were incubated in BS plus saponin for labeling with a second primary antibody or followed with secondary antibody. If another primary antibody was used, the cells were processed as above for total cell staining.
Confocal microscopy was performed using a laser scanning microscope (LSM 510; Carl Zeiss MicroImaging, Inc.), 40x water immersion lens (n = 1.5), at 25°C. Images were processed using Adobe Photoshop® (Adobe Systems, Inc.) version 7.0 software and Volocity (Improvision) version 2.0.1 software.
Antibodies
Antibodies used are as follows: C7, a mouse mAb to LDLR ectodomain (American Type Culture Collection); anti-T7, mouse mAb to T7 tag (Novagen); anti-cis Golgi, GM130 (Martin Lowe, University of Manchester, Manchester, UK); anti-Golgi, giantin (David Shima, Imperial Cancer Research Fund, London, UK), furin (Affinity BioReagents, Inc.); anti--adaptin were rabbit polyclonal (Margaret Robinson, University of Cambridge, Cambridge, UK); mouse monoclonal clone 88 (Transduction Labs); and clone 100/3 (Sigma-Aldrich). Anti-VSV-G, for IF, TK-G (Thomas Kreis), and for immunoprecipitation P5D4 (Thomas Kreis).
Pulse-chase biotinylation
Pulse-chase assays were performed as described previously (Matter et al., 1992). Samples were immunoprecipitated using P5D4 coupled to protein GSepharose beads (Zymed Laboratories). Biotinylated surface proteins were pulled down using neutravidin beads (Pierce Chemical Co.). Samples were analyzed by SDS-PAGE followed by Western blot. The gels were dried and quantitative autoradiography was performed using a PhosphorImager (Storm 860; Molecular Dynamics).
FACS® analysis
Flow cytometry was performed with a FACSCaliburTM with CellQuest software (Becton Dickinson) for acquisition and with FlowJo (TreeStar) for analysis. Secondary antibodies were antimouse phytoerythrin (Sigma-Aldrich).
Tfn uptake
MDCKT cells grown on coverslips and induced with 10 mM butyrate for 14 h. Cells were preincubated in serum-free media for 30 min at 37°C. Coverslips were inverted on a droplet of 100 µg/ml Tfn 594 or Tfn 488 (Molecular Probes, Inc.) in PBS on ice for 30 min, followed by incubation at 37°C for 22 min. Cells were processed for IF as described above.
Immuno-EM
EM was performed as described previously (Fölsch et al., 2001) using anti-GFP (CLONTECH Laboratories, Inc.) and anti-Alexa® 488 (Molecular Probes, Inc.).
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Acknowledgments |
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This work was supported by the National Institutes of Health (grants GM29765 and CA46128 to I. Mellman) and by the Ludwig Institute for Cancer Research.
Submitted: 8 July 2003
Accepted: 11 September 2003
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