Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Sep 1;121(Pt 17):2880-91.
doi: 10.1242/jcs.031641. Epub 2008 Aug 12.

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

Krystle S Spiczka  1 , Charles Yeaman
Affiliations

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

Krystle S Spiczka et al. J Cell Sci. .

Abstract

Changes in cellular behavior that cause epithelial cells to lose adhesiveness, acquire a motile invasive phenotype and metastasize to secondary sites are complex and poorly understood. Molecules that normally function to integrate adhesive spatial information with cytoskeleton dynamics and membrane trafficking probably serve important functions in cellular transformation. One such complex is the Exocyst, which is essential for targeted delivery of membrane and secretory proteins to specific plasma membrane sites to maintain epithelial cell polarity. Upon loss of cadherin-mediated adhesion in Dunning R3327-5'A prostate tumor cells, Exocyst localization shifts from lateral membranes to tips of protrusive membrane extensions. Here, it colocalizes and co-purifies with focal complex proteins that regulate membrane trafficking and cytoskeleton dynamics. These sites are the preferred destination of post-Golgi transport vesicles ferrying biosynthetic cargo, such as alpha(5)-integrin, which mediates adhesion of cells to the substratum, a process essential to cell motility. Interference with Exocyst activity impairs integrin delivery to plasma membrane and inhibits tumor cell motility and matrix invasiveness. Localization of Exocyst and, by extension, targeting of Exocyst-dependent cargo, is dependent on Ral GTPases, which control association between Sec5 and paxillin. Overexpression of Ral-uncoupled Sec5 mutants inhibited Exocyst interaction with paxillin in 5'A cells, as did RNAi-mediated reduction of either RalA or RalB. Reduction of neither GTPase significantly altered steady-state levels of assembled Exocyst in these cells, but did change the observed localization of Exocyst proteins.

PubMed Disclaimer

Figures

Figure 1
Figure 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 (“αSec8”) was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (B) Sub-confluent 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 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 point to 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. Arrowhead points to an accumulation of Exocyst within an actin-rich invadopodium. Scale bar = 20 μm.
Figure 2
Figure 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 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, Syntaxin3, Syntaxin4, Munc18c and exogenous GFP in 5′A cells. Sub-confluent cultures of R3327-5′A cells were cultured on type I collagen, and then processed for immunofluorescent staining with indicated antibodies, as described in Materials and Methods. Bound antibodies were detected with appropriate FITC or Texas Red-conjugated secondary antibodies, and epifluorescence images were obtained. Arrows point to accumulations of membrane trafficking components at the tips of pseudopods. Note that accumulations of Sec15, Exo84 and GFP were never observed within pseudopods. Scale bar = 20 μm.
Figure 3
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.
Figure 4
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).
Figure 5
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.
Figure 6
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.
Figure 7
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).
Figure 8
Figure 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 (“α-paxillin IP”) was assessed by SDS-PAGE followed by immunoblotting with specific antibodies.
Figure 9
Figure 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. Note that 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. Note that Exocyst proteins are concentrated in perinuclear compartments, similar to those observed in parental 5′A cells, when RalA expression is reduced. In contrast, these Exocyst subunits accumulate in large cytoplasmic vesicles in cells when RalB expression is reduced.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anitei M, Ifrim M, Ewart MA, Cowan AE, Carson JH, Bansal R, Pfeiffer SE. A role for Sec8 in oligodendrocyte morphological differentiation. J Cell Sci. 2006;119:807–18. - PubMed
    1. Bodemann BO, White MA. Ral GTPases and cancer: linchpin support of the tumorigenic platform. Nat Rev Cancer. 2008;8:133–40. - PubMed
    1. Boyd C, Hughes T, Pypaert M, Novick P. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J Cell Biol. 2004;167:889–901. - PMC - PubMed
    1. Bussemakers MJ, van Moorselaar RJ, Giroldi LA, Ichikawa T, Isaacs JT, Takeichi M, Debruyne FM, Schalken JA. Decreased expression of E-cadherin in the progression of rat prostatic cancer. Cancer Res. 1992;52:2916–22. - PubMed
    1. Camonis JH, White MA. Ral GTPases: corrupting the exocyst in cancer cells. Trends Cell Biol. 2005;15:327–32. - PubMed

Publication types

MeSH terms