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First published online 12 August 2008
doi: 10.1242/jcs.031641

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Ral-regulated interaction between Sec5 and paxillin targets Exocyst to focal complexes during cell migration

Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

View larger version (39K): [in this window] [in a new window]   Fig. 1. Exocyst expression, assembly and localization in non-metastatic and metastatic prostate tumor cells. (A) Dunning rat R3327-5'A (`A') and R3327-5'B (`B') cells were extracted in 1% Triton X-100. Extracts were subjected to immunoprecipitation with antibodies to Sec8. Presence of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 in equivalent amounts of whole cell extracts (`total') and precipitated immune complexes (`Sec8') was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (B) Subconfluent cultures of R3327-5'B, R3327-5'A or R3327-5'A cells stably expressing human E-cadherin were cultured on type I collagen-coated coverslips, and then processed for immunofluorescent staining with antibodies to Sec6 or Sec8, as described in the Materials and Methods. Samples were viewed with a Nikon Microphot-FX microscope (63x objective) and epifluorescent digital images were obtained using a Kodak DCS 760 digital camera. Arrows indicate accumulations of Exocyst proteins in protrusive extensions of R3327-5'A cells. (C) Sub-confluent cultures of R3327-5'A cells were cultured on Matrigel-coated coverslips, and then processed for immunofluorescent staining with phalloidin (to label f-actin) and antibodies to Sec6. Arrows indicate an accumulation of Exocyst within an actin-rich invadopodium. Scale bars: 20 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Exocyst subunits and SNARE proteins are enriched within protrusive cell extensions. (A) Dunning rat R3327-5'A prostatic tumor cells were seeded on 75 mm Transwell filters (3.0 µm pore size) and medium containing 10% FBS was added basolaterally to stimulate pseudopod extension, as described in the Materials and Methods. An enriched pseudopod fraction was obtained by removing cell bodies from the top of filters with a cotton swab. Indicated proteins were identified by immunoblotting with specific antibodies, and protein levels were quantified using a Molecular Dynamics Typhoon phosphorimager. To determine the fold enrichment of each protein within pseudopods, values were normalized to protein levels present in an equivalent amount of whole cell extract. (B) Distribution of endogenous Sec6, Sec8, Sec15, Exo84, syntaxin 3, syntaxin 4, Munc18c and exogenous GFP in 5'A cells. Subconfluent cultures of R3327-5'A cells were cultured on type I collagen, and then processed for immunofluorescent staining with indicated antibodies, as described in the Materials and Methods. Bound antibodies were detected with appropriate FITC or Texas Red-conjugated secondary antibodies, and epifluorescence images were obtained. Arrows and double-headed arrows indicate accumulation of membrane trafficking components at the tips of pseudopods. Accumulation of Sec15, Exo84 and GFP was never observed within pseudopods. Scale bar: 20 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Exocyst colocalizes 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 hours, 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 indicate tips of protrusive pseudopods, within which Sec6 and paxillin appear to colocalize. In lower panels, arrowheads indicate structures within pseudopods that stained with anti-paxillin antibodies, but not anti-Sec6 antibodies, and asterisks indicate structures that stained with anti-Sec6 antibodies but not anti-paxillin antibodies. (B) Distribution of endogenous Sec6, Git1, Nck1/2 and β-PIX in 5'A cells. Cells were cultured, fixed and permeabilized as described in the Materials and Methods. Sec6 distribution was compared with 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/argon laser with 488 nm (FITC) and 568 nm (Texas Red) laser lines. (C) Specificity of antibodies. 5'A cells were lysed and 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.

View larger version (65K): [in this window] [in a new window]   Fig. 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 the Materials and Methods. Fractions (0.5 ml) were collected and densities determined with a refractometer. Presence of Sec8, paxillin, NaK-ATPase 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 (Paxillin IP), normalized to total protein content, were assessed by SDS-PAGE followed by immunoblotting with specific antibodies to paxillin or Sec8. 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-Sec5RBD) 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 (-myc ip). Bands representing paxillin that co-immunoprecipitated with Myc-tagged Sec5 are indicated (asterisks).

View larger version (33K): [in this window] [in a new window]   Fig. 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 5-integrin (5-GFP) and morphological transport assays were performed as described in the 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. After 30 minutes 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 indicate TGN and arrows indicate examples of post-TGN transport intermediates. 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.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Exocyst is required for exocytosis of newly synthesized 5-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 the Materials and Methods. Sixty hours 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 5 integrin trafficking in prostate tumor cells. Delivery of newly synthesized 5-GFP to the surface of R3327-5'A cells was assessed as described in the 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 5-GFP bands, normalized to the mean of the total 5-GFP recovered in the initial immunoprecipitates.

View larger version (30K): [in this window] [in a new window]   Fig. 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 the Materials and 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 the Materials and 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).

View larger version (51K): [in this window] [in a new window]   Fig. 8. Ral GTPases are required for Exocyst association with paxillin in cells. (A) Localization of active Ral following wounding of prostatic tumor cells. Cells were transfected with X-Press-tagged Exo84 Ral-binding domain and monolayers were wounded by scratching. Active Ral GTPase localization was determined by immunofluorescence staining with anti-X-Press antibodies at indicated time points. (B) Co-immunoprecipitation of Exocyst and paxillin is dependent on Ral-binding capability of Sec5. Cells were transfected with plasmids encoding myc-tagged Sec5 [wild type (wt) or Ral-uncoupled mutants (T11A or R27E)]. Cells were extracted in 1% Triton X-100 and extracts were subjected to immunoprecipitation with antibodies to Sec8 or paxillin. Presence ectopic myc-Sec5 and paxillin in equivalent amounts of whole cell extracts (`lysate') and precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (C) RNAi-mediated reduction of RalA and RalB expression. R3327-5'A were infected with recombinant lentiviruses coding shRNAs specific for RalA or RalB. Stable clones of cells were selected in puromycin and assessed for RalA and RalB expression by immunoblotting with specific antibodies. (D) R3327-5'B, R3327-5'A or R3327-5'A cells expressing shRNAs targeting RalA or RalB were extracted in 1% Triton X-100. Extracts were subjected to immunoprecipitation with antibody to paxillin. Presence Sec5 and paxillin in equivalent amounts of whole cell extracts (`lysate') and precipitated immune complexes (`-paxillin IP') was assessed by SDS-PAGE followed by immunoblotting with specific antibodies.

View larger version (94K): [in this window] [in a new window]   Fig. 9. Ral GTPases are required for localization of Exocyst to protrusive cell extensions. (A) Morphology of R3327-5'A or R3327-5'A cells expressing shRNAs targeting RalA or RalB. RNAi-mediated reduction of either RalA or RalB suppressed polarized growth of protrusive cell extensions, and cells tended to grow as more tightly compacted colonies than control R3327-5'A prostate tumor cells. (B) Localization of Sec6 and Sec8 in prostate tumor cells is altered following reduction of RalA or RalB expression. R3327-5'A cells or R3327-5'A cells expressing shRNAs targeting RalA or RalB were fixed and labeled with antibodies against Sec6 or Sec8. Exocyst proteins are concentrated in perinuclear compartments, similar to those observed in parental 5'A cells, when RalA expression is reduced. By contrast, these Exocyst subunits accumulate in large cytoplasmic vesicles in cells when RalB expression is reduced.

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