RAL-REGULATED INTERACTION BETWEEN SEC5 AND PAXILLIN TARGETS EXOCYST TO FOCAL COMPLEXES DURING CELL MIGRATION

Exocyst is associated with paxillin-containing focal complexes within protrusive cellular extensions at the leading edge of migrating prostate tumor cells

If metastatic prostate tumor cells organize Exocyst assembly within pseudopods in order to target the delivery of cargo to these sites during cell migration, then the Exocyst should be enriched at sites of newly forming cell-substratum adhesions at the leading edge of cells, but not with older cell-substratum contacts, such as focal adhesions at the center or rear of cells. In order to promote directed motility of prostate tumor cells, cultures of 5′A cells were seeded at high density and experimental wounds were introduced by scratching the culture. Behavior of cells at the wound margin was then observed.

Paxillin is a molecular scaffold that organizes signaling proteins responsible for remodeling the plasma membrane and the actin cytoskeleton to orchestrate protrusive activity required for cell motility (Turner et al., 2001). In migrating fibroblasts, paxillin is associated with newly forming focal complexes at the leading edge, as well as with stable focal adhesions at the center and rear of cells. Immunofluorescent labeling studies were performed to determine whether the Exocyst co-localizes with components of paxillin-based signaling complexes. These included paxillin itself (Fig. 3A), the Arf6 GTPase-activating protein GIT1, the SH2/SH3 adaptor protein Nck1/2, and the Rac guanine nucleotide exchange factor β-PIX (Fig. 3B). That each of these proteins was expressed by 5′A cells and that antibodies to each were specific was confirmed by Western blot analysis (Fig. 3C).

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Figure 3

Exocyst co-localizes with focal complexes at pseudopod tips of migrating prostate tumor cells

(A) Distribution of endogenous Sec6 and paxillin in R3327-5′A prostatic tumor cells. Cells were seeded on fibronectin-coated glass coverslips for 18 hr, then were fixed with 2% paraformaldehyde, permeabilized with 1% Triton X-100, incubated with mouse anti-Sec6 (mAb 9H5) and rabbit anti-paxillin antibodies, then stained with FITC-conjugated goat anti-mouse and Texas Red-conjugated donkey anti-rabbit IgG. Epifluorescence images were obtained as described in Fig. 1. Arrows point to tips of protrusive pseudopods, within which Sec6 and paxillin appear to co-localize. In lower panels, arrowheads point to structures within pseudopods that stained with anti-paxillin antibodies, but not anti-Sec6 antibodies, and asterixes indicate structures that stained with anti-Sec6 antibodies but not anti-paxillin antibodies. (B) Distribution of endogenous Sec6, Git1, β-PIX and Nck1/2 in 5′A cells. Cells were cultured, fixed and permeabilized as described in Materials and Methods. Sec6 distribution was compared to that of Git1, β-PIX and Nck1/2. Sec6/Git1 and Sec6/β-PIX images were collected by epifluorescence microscopy. Sec6/Nck1/2 images were obtained with a Zeiss confocal laser-scanning microscope (63X objective) using a krypton/argo laser with 488 nm (FITC) and 568 nm (Texas Red) laser lines. (C) Specificity of antibodies. 5′A cells were lysed and approximately 1 μg of protein was loaded per lane onto a 10% SDS-PAGE gel. The proteins were transferred to Immobilon PVDF membranes and incubated with rabbit polyclonal antibodies to paxillin, GIT1 or Nck1/2, or mouse mAb to β-PIX. Blots were then probed with HRP-conjugated secondary antibodies and developed for ECL detection.

Paxillin, GIT1, Nck1/2 and β-PIX were all enriched at pseudopods tips, similar to the Exocyst (Fig. 3). Each of these proteins was also present at juxtanuclear sites. Close inspection of cells by confocal microscopy revealed that Sec6 and paxillin were co-localized at distal tips of pseudopods at the leading edge of migrating cells at the wound margin (Fig. 3A, lower panels, arrows), but that behind these sites, more proximal to cell bodies, the two proteins had distinct distributions (Fig. 3A, lower panels, arrowheads and asterixes). Importantly, paxillin was also enriched within adhesion sites elsewhere in the cell, but Sec6 was not enriched at these sites. Paxillin-associated signaling proteins were, like Sec6, enriched within pseudopods tips but did not appear to accumulate within other paxillin-positive structures (Fig. 3B). Therefore, these results indicate that Exocyst is enriched at sites containing newly forming cell-substrate focal complexes at the leading edge of migrating cells, but is absent from older contact sites elsewhere in cells.

Paxillin, β-PIX, GIT1 and Nck1/2 co-fractionate with Sec8 in density gradients of post-nuclear supernatant obtained from mechanically homogenized 5′A prostate tumor cells (Fig. 4A). Analysis of gradient fractions by western blotting with specific antibodies reveals a peak of Sec8 in fractions corresponding to a density (δ) of ~1.15 – 1.17 g/ml (Fig. 4A, peak “A”). Thus, Sec8 is associated with membranes that have a higher density than the bulk of plasma membrane, as defined by the presence of sodium-potassium ATPase (Fig. 4A, NaK-ATPase, peak δ ~1.10 – 1.13 g/ml). A fraction of paxillin was recovered in the Sec8-containing peak, although a second peak of paxillin was recovered in higher density fractions that did not contain a peak of Sec8 (Fig. 4A, peak “B”). Importantly, β-PIX and GIT1 were primarily recovered within the Sec8-containing fractions, and not with the higher density paxillin-containing fractions. The SH2/SH3 adaptor Nck1/2 was found to co-fractionate with both paxillin-containing peaks, but a slightly larger (~47 kDa) protein that was detected by anti-Nck1/2 antibodies was present exclusively in fractions that contained Sec8. This band appears to represent a phosphorylated form of Nck1/2, because treatment of fractions with alkaline phosphatase caused it to disappear (data not shown). These results indicate that Sec8 co-fractionates in density gradients with membranes containing paxillin and associated proteins involved in regulating vesicle trafficking and cytoskeleton dynamics.

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Figure 4

Exocyst is associated with paxillin-containing complexes within pseudopods of migrating prostate tumor cells

(A) Fractionation of R3327-5′A cells in iodixanol gradients. 5′A cells were homogenized in a ball bearing cell cracker. Post-nuclear supernatant was fractionated by isopycnic centrifugation through five-step iodixanol gradients, as described in Materials and Methods. Fractions (0.5 ml) were collected and densities determined with a refractometer. Presence of Sec8, paxillin, NaK-ATPase α subunit, β-PIX, GIT1 and Nck1/2 in gradient fractions was assayed by SDS-PAGE followed by immunoblotting with specific antibodies. Protein levels were quantified using a Molecular Dynamics Phosphorimager. Fractions corresponding to peak levels of NaK-ATPase, Sec8 and paxillin are labeled “plasma membrane”, “A” and “B”, respectively. (B) Co-immunoprecipitation of Sec8 with paxillin from isolated 5′A pseudopods. 5′A cells were cultured on 75 mm Transwell filters (3 μm pores), and induced to extend pseudopods, as described for Fig. 2. Whole cells (“wc”), isolated pseudopods (“p”) or cell bodies (“cb”) were isolated in CSK buffer after rubbing either the top or bottom of filters with a cotton swab, as appropriate. Extracts (“total”) and precipitated immune complexes (“αPaxillin IP”), normalized to total protein content, were assessed by SDS-PAGE followed by immunoblotting with specific antibodies to paxillin or Sec8. Note that Sec8 co-precipitates with paxillin immune complexes, but only from pseudopods and not cell bodies. (C) Sec5 binds paxillin in vitro. Plasmids encoding paxillin and/or myc-Sec5 or Sec5 lacking its Ral-binding domain (myc-Sec5ΔRBD) were used to prime coupled transcription and translation reactions in the presence of [³⁵S]methionine/cysteine. Aliquots of translation products were assessed directly (“total”) or after immunoprecipitation with anti-myc antibodies (“α-myc ip”). Bands representing paxillin that co-immunoprecipitated with Myc-tagged Sec5 are indicated (asterixes).

Results of density gradient fractionation suggest that fractions containing the peak of Sec8 (Fig. 4A, peak “A”) may contain pseudopod tip complexes, whereas fractions containing the peak of paxillin (Fig. 4A, peak “B”) may contain older focal adhesion complexes that do not contain Exocyst proteins. To confirm the prediction that Exocyst associates preferentially with focal complexes at the leading edge of migrating cells, paxillin-containing protein complexes were immunoprecipitated from cell bodies or pseudopods isolated from 5′A cells induced to undergo frustrated chemotaxis, as described for Fig. 2. This analysis confirmed that Exocyst complexes were only associated with paxillin-containing complexes isolated from pseudopods, and not with more abundant paxillin complexes associated with other cellular structures (Fig. 4B).

To determine whether the Exocyst directly binds paxillin, and to identify which subunit is responsible for this interaction, each individual Exocyst subunit was expressed together with paxillin in coupled in vitro transcription/translation reactions. This analysis revealed a direct interaction between Sec5 and paxillin (Fig. 4C). None of the other Exocyst subunits associated with paxillin in this assay (data not shown). This interaction was independent of the amino terminal Ral-binding domain of Sec5, indicating that paxillin binds a different part of Sec5 than that involved in binding Ral GTPases (Fig. 4C).

Biosynthetic cargo is delivered to Exocyst-containing pseudopods

Morphological transport assays were performed to determine whether exocytic cargo is preferentially targeted to Exocyst-enriched sites in pseudopods. For these studies, cells were transfected with plasmids encoding GFP-tagged forms of either α₅-integrin (α₅-GFP) or a temperature-sensitive mutant of the vesicular stomatitis virus (VSV) G protein (tsG-GFP). The latter protein is a convenient marker of exocytosis because its transport through the secretory pathway can be manipulated by shifting the temperature at which cells are cultured. Following transfection, cells were incubated at 39.5 °C for 4 hours to accumulate tsG-GFP in the endoplasmic reticulum, then shifted to 19 °C for two hours to allow the protein to accumulate within the trans-Golgi network (TGN). Finally, cells were shifted to 32 °C for various lengths of time to permit protein trafficking from the TGN to the plasma membrane. In cells fixed after incubation at 32°C for 30 minutes, much of the GFP-tagged cargo remained in perinuclear compartments (Fig. 5A, arrowheads). In addition, post-Golgi transport intermediates containing tsG-GFP or α₅-GFP were observed in the cytoplasm at some distance from the perinuclear vicinity, and these appeared to be enriched in pseudopods compared with other parts of the cell (Fig. 5A, arrows).

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Figure 5

Polarized trafficking of biosynthetic cargo to pseudopods in prostate tumor cells

R3327-5′A cells were transfected with plasmids encoding GFP-VSVG (tsG-GFP) or GFP α₅-integrin (α₅-GFP) and morphological transport assays were performed as described in Materials and Methods. Following accumulation of cargo proteins in the TGN, cultures were shifted to 32 °C either in the absence (A) or presence (B) of 0.5% tannic acid for various lengths of time to facilitate the trafficking of accumulated GFP-fusion proteins from the TGN to the plasma membrane. Following 30 min at 32 °C (A) or indicated times (B), cells were fixed with 2 % paraformaldehyde, permeabilized and stained with anti-Sec8 mAb, which was detected using a Texas Red-conjugated secondary antibody. Epifluorescence images were obtained as described in Fig. 1. Arrowheads point to TGN and arrows point to examples of post-TGN transport intermediates. Note that more post-TGN transport vesicles appear to be delivered to pseudopods than to other parts of the cell, even when the TGN is located on the opposite side of the nucleus from the pseudopod. In tannic acid pre-fixed samples (B), pseudopods accumulate transport vesicles, but with prolonged incubation vesicles also begin to accumulate within the cell body.

Upon docking and fusion with the plasma membrane, it is no longer possible to observe accumulation of GFP-labeled transport vesicles in the cytoplasm. It could be argued, therefore, that post-Golgi transport intermediates only appear to be targeted to pseudopods because they have farther to travel than do intermediates that dock and fuse within the cell body. Therefore, terminal docking and fusion events were impaired by application of the non-permeable fixative tannic acid to cells in order to more definitively resolve whether exocytosis was polarized towards pseudopods. Tannic acid crosslinks proteins at the plasma membrane, thereby “freezing” the vesicle fusion machinery (Polishchuk et al., 2004). However, cytoplasmic events, including post-Golgi vesicle budding and transport to the cell periphery proceed unimpeded in the presence of tannic acid for at least one hour. Under these conditions, a clear accumulation of both tsG-GFP and α₅-GFP-containing transport intermediates was observed within pseudopods (Fig. 5B). After prolonged incubation at 32 °C, vesicles began to accumulate within the cell body, indicating either that pseudopods had exceeded their capacity for vesicles or that transport machinery was failing due to extended exposure to tannic acid. Nevertheless, these results indicate that exocytosis is polarized towards Exocyst-enriched tips of pseudopods in 5′A prostate tumor cells.

Exocyst assembly within pseudopods is required for exocytosis and invasive cell motility

To determine whether the Exocyst is required for biosynthetic protein trafficking in metastatic prostate tumor cells, the efficiency of delivery of newly synthesized α₅-integrin to the surface of 5′A cells that had normal or reduced levels of Sec5 or Sec6 was compared. Cells were transfected with siRNAs specific for Sec5 or Sec6 or a control siRNA. 24 hr later, cells were transfected with plasmid encoding α₅-GFP, then returned to the incubator for another 24 hr. On the day of the experiment (48 hr after siRNA transfection), one set of cultures was lysed and analyzed for expression of Sec5, Sec6 or a loading control (β-PIX) by Western blotting (Fig. 6A), and another set was used to quantify α₅-GFP trafficking (Fig. 6B). Triplicate wells containing either control or Exocyst knock-down cells were subjected to pulse-chase and surface biotinylation analysis to quantify the amount of newly synthesized α₅-GFP that arrived at the surface during a one hour delivery period. This revealed that the efficiency of delivery of newly synthesized α₅-GFP to the plasma membrane of 5′A cells was significantly reduced following RNAi-mediated reduction of Sec5 or Sec6 expression. The amount of α₅-integrin that was synthesized in control and experimental cultures was similar (data not shown), but the fraction that was inserted into the plasma membrane of cells with reduced Sec5 or Sec6 levels was reduced to approximately 18 – 22 % of the levels observed in mock-transfected cells or in cells transfected with control siRNA. Therefore, Exocyst function is required for trafficking of newly synthesized α₅-integrin from the TGN to the plasma membrane of prostate tumor cells.

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Figure 6

Exocyst is required for exocytosis of newly synthesized α5-integrin in prostate tumor cells

(A) RNAi mediated reduction of Sec5 and Sec6 expression. R3327-5′A were transfected with either nothing (“mock”) or with siRNAs targeting Sec5, Sec6 or a control non-targeting siRNA (“control”), as described in Materials & Methods. 60 hr post-transfection, cells were lysed and lysates were analyzed by SDS-PAGE and immunoblotting for Sec5, Sec6 and β-PIX. Protein levels were quantified using a Molecular Dynamics Typhoon phosphorimager. (B) Metabolic pulse-chase and surface biotinylation analysis of α5 integrin trafficking in prostate tumor cells. Delivery of newly synthesized α5-GFP to the surface of R3327-5′A cells was assessed as described in Materials and Methods. Experiments were performed twice, each time with triplicate wells of cells. Relative surface delivery was defined as the mean signal obtained from three replicate biotinylated α5-GFP bands, normalized to the mean of the total α5-GFP recovered in the initial immunoprecipitates.

Because Exocyst complexes are enriched within pseudopods and invadopodia and are required for exocytosis of α₅-GFP-laden transport vesicles, it is reasonable to expect that Exocyst assembly within pseudopods and invadopodia is required for invasive cell motility. Wound healing experiments were performed initially, to determine whether Exocyst assembly is required for directed motility of 5′A prostate tumor cells. Following RNAi-mediated reduction of either Sec5 or Sec6, cells were significantly impaired in their ability to close wounds relative to control cells (Fig. 7A). 24 hr after wounding monolayers, control monolayers had completely healed, whereas wounded monolayers of cells with reduced Sec5 or Sec6 levels had closed wounds to only ~ 35–40% of the controls. Scattered individual cells were observed within the wounds in these cultures, but coordinated migration of the population was impaired following Sec5 or Sec6 knockdown.

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Figure 7

Ral-coupled Exocyst activity is required for invasive motility of prostate tumor cells

(A) Wound healing assay. Confluent monolayers of R3327-5′A cells, transfected with siRNAs specific for Sec5, Sec6 or a control non-targeting siRNA, were experimentally wounded by scratching, and analysis was performed as described in Materials & Methods. (B) Matrigel invasion assay. Non-metastatic R3327-5′B cells, or metastatic R3327-5′A cells transfected with indicated siRNAs and rescue constructs, were seeded on Matrigel-coated Transwell filters. Invasion assays were performed as described in Materials & Methods. (C) Sec5 expression analysis. 5′A prostate tumor cells transfected with indicated siRNAs and/or rescue constructs, were extracted and analyzed by SDS-PAGE and immunoblotting with antibodies to Sec5 (endogenous and ectopic proteins detected) or c-myc (ectopic Sec5 detected).

A hallmark of metastatic tumor cells is their ability to degrade basal laminae and subjacent connective tissue, enter the bloodstream and migrate to distant sites in the body. To determine whether Exocyst function is required for the invasive motility of 5′A prostate tumor cells, the ability of cells to migrate across Matrigel-coated filters was assayed (Fig. 7B). Non-metastatic 5′B prostate tumor cells were not able to degrade Matrigel and so remained on the upper surface of filters during invasion assays. In contrast, a significant number of metastatic 5′A cells were able to degrade Matrigel and migrate across filters during a 24 hr incubation (Fig. 7B). In 5′A cells transfected with Sec5 or Sec6 siRNAs, but not with control siRNA, invasiveness was impaired and cells remained in the upper chamber, similar to non-metastatic 5′B cells (Fig. 7B). Co-transfection of cells with Sec5 siRNA and a plasmid encoding myc-tagged Sec5 harboring a silent mutation to render the mRNA resistant to RNAi-mediated silencing restored Sec5 expression (Fig. 7C) and rescued ECM invasiveness to levels comparable to control 5′A cells (Fig. 7B).

Cell Culture Methodology

Dunning rat prostate tumor cell lines (R3327-5′A and R3327-5′B) were maintained in high glucose Dulbecco’s modified Eagle’s media (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and kanamycin. Plasmids were transfected using Lipofectamine2000 reagent (Life Technologies) according to the manufacturer’s instructions. siRNAs were transfected at 160 nM using Oligofectamine (Life Technologies) according to the manufacturer’s instructions. Lentivirus production in 293FT packaging cells followed established protocols (The RNAi Consortium). Stable knockdown of RalA and RalB was achieved by lentiviral infection of R3327-5′A cells and selection in 5 μg/ml puromycin.

Immunofluorescent Staining

Cells were fixed in 4% paraformaldehyde for 30 min, then permeabilized by incubation 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 and protease inhibitors (1 mM pefabloc, and 10 μg/ml each of aprotinin, antipain, leupeptin, pepstatin A) (CSK buffer). Antibodies were diluted in blocking buffer (Ringer’s saline (154 mM NaCl, 1.8 mM Ca²⁺, 7.2 mM KCl, 10 mM Hepes, pH 7.4) containing 0.2 % BSA, 0.5% normal goat serum and 0.5% normal donkey serum) and applied to cells for 2 h at 4°C. After five washes in blocking buffer, FITC and Texas Red conjugated secondary antibodies, diluted 1:200, were applied for 1 h at 4°C. Coverslips and filters were washed five times and mounted in VectaShield (Vector Laboratories, Burlingame, CA). Samples were viewed with either a Nikon Microphot-FX microscope (63X or 100X objectives) or a Zeiss 510 confocal laser scanning microscope (63X objective) using krypton/argon laser with 488 nm (FITC, GFP) and 543 nm (Texas Red) laser lines, as noted in figure legends. Digital images of data collected from the Nikon Microphot-FX microscope were obtained with a Kodak DCS 760 digital camera.

Morphological assay for polarized trafficking of α₅-GFP and ts-G-GFP

R3327-5′A cells were transfected with plasmids encoding either α₅-GFP or ts-G-GFP. To accumulate ts-G-GFP in the TGN, cells were suspended with trypsin-EDTA 24 hr after transfection, re-plated on collagen-coated coverslips and incubated at 40°C for 5 hr to accumulate ts-G-GFP in the endoplasmic reticulum. Cultures were shifted to 32°C for 7.5 min to facilitate protein folding, and subsequently transferred to 19°C for 2 hr to accumulate protein in the TGN. To accumulate α₅-GFP in the TGN, cells were suspended with trypsin-EDTA 24 hr after transfection, re-plated on collagen-coated coverslips and incubated at 37°C for 1 hr then transferred to 19°C for 4 hr. Trafficking of protein from the TGN to plasma membrane was initiated by shifting cultures from 19°C to 32°C. In some cases, cultures were treated with 0.5 % (w/v) tannic acid prior to and during the final 32°C incubation period to inhibit vesicle fusion with the plasma membrane (Polishchuk et al., 2004). At indicated time points, cultures were fixed and labeled with antibodies to Sec8 followed by Texas Red anti-mouse secondary antibody.

Gel Electrophoresis and Immunoblotting

Protein samples were incubated in SDS-PAGE sample buffer for 10 min at 65°C before separation in 7.5%, 10% or 12.5% SDS polyacrylamide gels. Proteins were electrophoretically transferred from gels to Immobilon PVDF membrane (Millipore Corp., Bedford, MA). Blots were blocked in Blotto (5% nonfat dry milk, 0.1% sodium azide in 150 mM NaCl, 10 mM TrisHCl, pH 7.5 [TBS]) overnight at 4°C. Primary antibodies were incubated with blots at room temperature for 1 h. After five washes, 10 min each, in TBS containing 0.1% Tween-20, the blots were incubated with ¹²⁵I-labeled goat anti-mouse or goat anti-rabbit secondary antibody (Amersham) for 1 h at room temperature. Blots were washed as above and exposed to phosphorimager screens. The amount of labeled antibody bound to the blots was determined directly using a Phosphorimager (Typhoon, Molecular Dynamics, Sunnyvale, CA) and ImageQuant software (version 1.2, Molecular Dynamics, Sunnyvale, CA).

Cell Fractionation in Iodixanol Gradients

Cells were homogenized in isotonic sucrose buffer (0.25 M sucrose in 20 mM HEPES-KOH, pH 7.2, 90 mM KOAc, 2 mM Mg(OAc)₂, and protease inhibitors) by repeated passage through a ball bearing homogenizer (Varian Physics, Stanford University). Separation of different membrane compartments was achieved by centrifugation in five-step 10–15–20–25–30% (wt/vol) iodixanol gradients. Briefly, post-nuclear supernatant was mixed with Opti-Prep (60% (wt/vol) iodixanol, Nycomed, Oslo, Norway) and homogenization buffer to generate a solution containing 30% iodixanol. This was overlayed in centrifuge tubes with equal volumes of 25%, 20%, 15%, and 10% iodixanol, and samples were centrifuged at 350,000 × g for 3 h at 4°C in a Beckman NVT65 rotor. Fractions (0.5 ml) were collected, refractive indices were read, and proteins were separated by SDS-PAGE. Proteins were transferred from gels to Immobilon P membranes for immunoblotting as described above.

Isolation of pseudopods

Pseudopod isolation was performed similar to methods described previously (Cho and Klemke, 2002). R3327-5′A cells were incubated for 24 hr in serum-free DMEM containing 0.5 % (w/v) BSA and antibiotics. Cells were suspended and seeded at a density of 1.5 × 10⁷ cells/filter on Transwell filters (# 3420, 75 mm diameter, 3.0 μm pore size). Following attachment at 37°C for 2 hr in serum-free medium, complete medium containing 10% FBS was applied to the basal chamber to stimulate chemotaxis. Cultures were returned to 37°C and incubated for 4 hr to allow pseudopod growth. Cell bodies were manually removed from the tops of filters by gentle scrubbing with a cotton swab, leaving the detached pseudopods adherent to the bottoms of filters. For analysis of total cellular protein, cell bodies were not removed. Lastly, intact cells or isolated pseudopods were solubilized in extraction buffer (CSK with protease inhibitors) and protein content was quantified using a micro BCA assay (Pierce).

Immunoprecipitation

Whole cells, pseudopods or cell bodies were extracted in CSK buffer for 30 min at 4°C. Extracts were precleared with 5 μl of nonimmune serum and 50 μl Staphylococcus aureus cells (Pansorbin; Calbiochem Novabiochem, La Jolla, CA) for 1 hr at 4°C. For Sec8 immunoprecipitation, mAbs 2E12, 5C3 and 10C2 were covalently cross-linked to Protein A Sepharose beads (Pharmacia LKB Nuclear, Gaithersburg, MD) with dimethyl pimelimidate (DMP), and 20 μl of immunoadsorbant was used per immunoprecipitation. Immunoprecipitation of paxillin was performed with a specific rabbit polyclonal antibody (5 μg per sample) pre-bound to Protein A Sepharose beads. Immunoadsorbants were incubated with pre-cleared cell extracts for 2 h at 4°C, then washed under stringent conditions and prepared for SDS-PAGE as described previously (Yeaman et al., 2004).

In Vitro Transcription/Translation Analysis

Plasmids encoding individual myc-tagged Exocyst subunits (in pGBKT7 vectors) were added singly or together with plasmid encoding paxillin (in pcDNA 3.1) to TNT Quick Coupled Transcription/Translation Systems (Promega Corp.). Reactions were incubated in the presence of [³⁵S]methionine/cysteine (Perkin-Elmer), according to manufacturer’s instructions. Aliquots of total translated product or anti-myc immunoprecipitated material were analyzed by SDS-PAGE and autoradiography.

Metabolic Pulse-Chase and Surface Biotinylation Analysis

R3327-5′A cells were transfected with siRNAs specific for Sec5 or Sec6 or a control siRNA. 24 hr later, cells were transfected with plasmid encoding α₅-GFP and returned to the incubator. The following morning, cells were suspended with trypsin and split into four identical replicate cultures and returned to the incubator for 12 – 18 hr. On the day of the experiment (~60 hr after siRNA transfection), one set of cultures was lysed and analyzed for expression of Sec5, Sec6 or a loading control (β-PIX) by Western blotting, and the remaining cultures were used to quantify α₅-GFP trafficking. Triplicate wells containing either control or Exocyst knock-down cells were incubated in methionine/cysteine-free culture medium to starve them for 30 min, then pulse-labeled for 30 min in the same medium supplemented with 1 mCi/ml [³⁵S]methionine/cysteine. Following the labeling period, cultures were washed three times in chase medium (complete DMEM supplemented with 5-fold excess methionine and cysteine), and returned to the incubator for one hour in the same medium. At the end of the chase incubation, cultures were placed on ice, washed with ice-cold Ringer’s saline, and labeled with Sulfo-NHS-SS-Biotin (Pierce). Following extensive washes in quenching buffer (TBS containing 50 mM NH₄Cl and 0.5 % BSA), cells were lysed and pre-cleared as described above, and α₅-GFP was immunoprecipitated with anti-GFP antibodies. The biotinylated (eg surface-exposed) cohort of α₅-GFP was subsequently recovered from total immunoprecipitates by avidin precipitation. Samples were analyzed by SDS-PAGE and bands were quantified with a phosphorimager following fluorographic enhancement of gels with Amplify solution (Amersham). Relative surface delivery of α₅-GFP was defined as the mean signal obtained from three replicate biotinylated α₅-GFP bands, normalized to the mean of the total α₅-GFP recovered in initial immunoprecipitates.

Wound-healing assays

R3327-5′A cells were transfected with siRNAs specific for Sec5, Sec6 or a control siRNA using Oligofectamine. 72 hr post-transfection, confluent cell monolayers were experimentally wounded by scratching with a pipette tip, rinsed with Ringer’s saline, and fed with fresh medium containing 10% serum. Analysis of wounds was performed by photographing several areas of each wound at multiple time points over a 48 hr period using a Nikon TE300 microscope equipped with a Nikon Coopix 5000 camera. Areas of wound closure were quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).

This work was supported by grants from the National Institutes of Health (GM067002) and the U.S. Department of Defense (DAMD 17-03-1-0187). Brian Leper, Melinda Schwarz and Hsiang Wen are gratefully acknowledged for their technical assistance in generating and characterizing reagents and performing these studies.