doi: 10.1091/mbc.e08-02-0199. Epub 2009 Apr 22.
Vimentin regulates scribble activity by protecting it from proteasomal degradation
Affiliations
- PMID:19386766
- PMCID: PMC2695792
- DOI: 10.1091/mbc.e08-02-0199
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Vimentin regulates scribble activity by protecting it from proteasomal degradation
Dominic C Y Phua et al. Mol Biol Cell.2009 Jun.
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Abstract
Scribble (Scrib), Discs large, and Lethal giant larvae form a protein complex that regulates different aspects of cell polarization, including apical-basal asymmetry in epithelial cells and anterior-posterior polarity in migrating cells. Here, we show that Scrib interacts with the intermediate filament cytoskeleton in epithelial Madin-Darby canine kidney (MDCK) cells and endothelial human umbilical vein endothelial cells. Scrib binds vimentin via its postsynaptic density 95/disc-large/zona occludens domains and in MDCK cells redistributes from filaments to the plasma membrane during the establishment of cell-cell contacts. RNA interference-mediated silencing of Scrib, vimentin, or both in MDCK cells results in defects in the polarization of the Golgi apparatus during cell migration. Concomitantly, wound healing is delayed due to the loss of directional movement. Furthermore, cell aggregation is dependent on both Scrib and vimentin. The similar phenotypes observed after silencing either Scrib or vimentin support a coordinated role for the two proteins in cell migration and aggregation. Interestingly, silencing of vimentin leads to an increased proteasomal degradation of Scrib. Thus, the upregulation of vimentin expression during epithelial to mesenchymal transitions may stabilize Scrib to promote directed cell migration.
Figures
Scrib localizes to intermediate filaments. (A–D) Localization of endogenous Scrib to vimentin IFs in MDCK cells and HUVECs. A few coverslips were placed into a 10-cm dish and MDCK cells (A and B) were seeded at 1 × 106 (A; Sparse) or 1 × 107 (B; Confluent) cells and grown for 2 d. HUVECs (C and D) were seeded at 1.6 × 105 (C; Sparse) or 6.5 × 105 (D; Confluent) cells per 10-cm dish and grown for 4 d. Coverslips were then removed and used for immunofluorescence microscopy; the remaining culture in the dish was imaged by phase contrast to assess cell confluence and then processed for biochemical analysis (see Figure 2A, a and c). Scrib (A–D, a, green) and vimentin (A–D, b, red) were immunostained. Yellow in the merged images (A–D, c) indicates colocalization. In sparse cultures of both cell types, punctate staining of endogenous Scrib can be observed along vimentin filaments emanating from the perinuclear region toward the cell periphery (arrowheads in magnification of blue inset in A and C, d–f). In addition, in MDCK cells, Scrib can also be detected on vimentin filaments peripheral to the plasma membrane (arrowheads in magnification of yellow inset in A, g–i). In HUVECs, both Scrib and nonfilamentous vimentin colocalize at the plasma membrane as punctate staining in what seem to be membrane protrusions (arrowheads in magnification of yellow inset in C, g–i). In confluent cultures, Scrib is concentrated at contact sites and present on vimentin filaments in the cell periphery of MDCK cells (arrowheads in magnification of yellow inset in B, d–f). In HUVECs, the Scrib positive contact sites colocalize with nonfilamentous vimentin or short filament staining (arrowheads in magnification of yellow inset in D, d–f). Note the presence of short vimentin filaments in the cell periphery. (E) Epitope-tagged hScrib colocalizes with vimentin and keratin 18. A few coverslips were placed into a 10-cm dish and MDCK cells expressing EGFP-hScrib were seeded at 1 × 106 cells per 10-cm dish and grown for 2 d to obtain sparse cultures. Coverslips were then removed and used for immunofluorescence microscopy; the remaining culture in the dish was imaged by phase contrast to assess cell confluence and then processed for biochemical analysis (see Figure 2Ab). EGFP-hScrib (a and d, green) and vimentin (b, red) or keratin 18 (e, red) were visualized in sparse MDCK cells expressing EGFP-hScrib. Yellow in the merged images (c and f) indicates colocalization of hScrib with the IF. (F and G) Relocalization of EGFP-hScrib, vimentin, and keratins during formation of polarized MDCK cell monolayers grown on permeable supports. EGFP-hScrib expressing MDCK cells were seeded at 3 × 105 cells per 12-mm-diameter permeable support insert and grown for 1 d (Sparse) or 7 d (Confluent). EGFP-hScrib (F and G, a and d, green) and vimentin (F and G, b, red color) or keratin 18 (F and G, e, red) were visualized by confocal fluorescence microscopy in sparse (F) or polarized (G) MDCK cell cultures. Blue lines indicate the location of the horizontal confocal section, and red and green lines indicate the site of vertical confocal sections along the apical–basal axis of the monolayer. Yellow in the merged images indicate colocalization (F and G, c and f). Note the extensive colocalization of EGFP-hScrib with vimentin and keratins in nonpolarized cells. In polarized cells, vimentin and keratin 18 accumulate at the apical pole, whereas EGFP-hScrib is present on the lateral membrane and only shows minimal overlap with IFs at the apical end of the lateral membrane.
Scrib associates with intermediate filaments via its PDZ domains. (A) Scrib interacts with vimentin in MDCK cells and HUVECs at various culture confluences. Control MDCK cells (a) or cells expressing EGFP-hScrib (b) were seeded at 1 × 106, 2 × 106, 5 × 106, or 1 × 107 cells per 10-cm dish and grown for 2 d to reach different confluences (top, phase contrast images). Vimentin was immunoprecipitated from harvested lysates. Associated endogenous (a) or exogenous (b) Scrib was detected by Western blot. Precipitation with an antibody to β-catenin served as a negative control. An aliquot of the cell lysate (5%) was directly analyzed by Western blot to monitor Scrib and vimentin expression levels (Input). GAPDH was detected to monitor for equal cell lysate loading. HUVECs were seeded at 1.6 × 105 (Sparse) or 6.5 × 105 (Confluent) cells per 10-cm dish and grown for 4 d (top, phase contrast images). Scrib was immunoprecipitated from harvested lysates. Associated vimentin was detected by Western blot. Precipitation with normal IgG served as a negative control (c). An aliquot of the cell lysate (5%) was directly analyzed by Western blot to monitor Scrib and vimentin expression levels (Input). (B) Scrib associates directly with both nonpolymerized or polymerized vimentin in vitro. Vimentin assembly was monitored through fractionation by ultracentrifugation. Fractions were subjected to Western blot and vimentin visualized as nonpolymerized (NonPolymer) or polymerized (Polymer) forms in the supernatant or pellet, respectively (a). In vitro-translated HA-mScrib cosedimented with vimentin only when applied to polymerized vimentin but not alone (b). HA-mScrib also associated with both polymerized (c) and nonpolymerized (d) vimentin in coimmunoprecipitation assays. Note that even in the presence of a large excess of normal IgG (d), little if any Scrib is found in the precipitate. (C) Schematic diagram of EGFP-hScrib deletion mutants. Full-length human Scrib was either tagged at the N terminus (GFP-Scrib) or C terminus (Scrib-GFP) with EGFP. ΔCter lacks the region C-terminal to the PDZ domains, which is contained in the Cter construct. LRR and PDZ encode the N-terminal LRR-LAPSD or the C-terminal four PDZ domains, respectively. 4PDZ contains the PDZ domains only. aa, amino acids. (D) Scrib associates with IFs via the region containing the four PDZ domains. EGFP-hScrib WT, LRR, and PDZ were immunoprecipitated from sparse MDCK cells and associated vimentin and keratin 18 as detected by Western blot analysis (a). The detection of ZO-2 served as a positive control for the interaction with hScrib WT or PDZ and as a negative control for the interaction with LRR (Métaiset al., 2005). An aliquot (5%) of the cell lysate was directly subjected to Western blot analysis to monitor the expression levels of vimentin, keratin 18, and ZO-2 (Input) (b). Scrib directly binds to vimentin via its PDZ domains. Purified in vitro-polymerized (c) or nonpolymerized (d) vimentin and in vitro-translated HA-mScrib LRR or HA-mScrib PDZ were combined and incubated. Vimentin was then immunoprecipitated and vimentin or HA-mScrib detected by Western blot. Normal IgG served as a negative control. (E) hScrib PDZ localizes to vimentin filaments in sparse MDCK cells. The different hScrib constructs tagged with EGFP were expressed in MDCK cells and visualized by Western blot analysis (a) or fluorescence microscopy (b–o). Note that both N- and C-terminally tagged Scrib (b and c) and only constructs containing the PDZ domains (d–f, j–l, and m–o) show extensive filamentous localization.
Scrib associates with intermediate filaments via its PDZ domains. (A) Scrib interacts with vimentin in MDCK cells and HUVECs at various culture confluences. Control MDCK cells (a) or cells expressing EGFP-hScrib (b) were seeded at 1 × 106, 2 × 106, 5 × 106, or 1 × 107 cells per 10-cm dish and grown for 2 d to reach different confluences (top, phase contrast images). Vimentin was immunoprecipitated from harvested lysates. Associated endogenous (a) or exogenous (b) Scrib was detected by Western blot. Precipitation with an antibody to β-catenin served as a negative control. An aliquot of the cell lysate (5%) was directly analyzed by Western blot to monitor Scrib and vimentin expression levels (Input). GAPDH was detected to monitor for equal cell lysate loading. HUVECs were seeded at 1.6 × 105 (Sparse) or 6.5 × 105 (Confluent) cells per 10-cm dish and grown for 4 d (top, phase contrast images). Scrib was immunoprecipitated from harvested lysates. Associated vimentin was detected by Western blot. Precipitation with normal IgG served as a negative control (c). An aliquot of the cell lysate (5%) was directly analyzed by Western blot to monitor Scrib and vimentin expression levels (Input). (B) Scrib associates directly with both nonpolymerized or polymerized vimentin in vitro. Vimentin assembly was monitored through fractionation by ultracentrifugation. Fractions were subjected to Western blot and vimentin visualized as nonpolymerized (NonPolymer) or polymerized (Polymer) forms in the supernatant or pellet, respectively (a). In vitro-translated HA-mScrib cosedimented with vimentin only when applied to polymerized vimentin but not alone (b). HA-mScrib also associated with both polymerized (c) and nonpolymerized (d) vimentin in coimmunoprecipitation assays. Note that even in the presence of a large excess of normal IgG (d), little if any Scrib is found in the precipitate. (C) Schematic diagram of EGFP-hScrib deletion mutants. Full-length human Scrib was either tagged at the N terminus (GFP-Scrib) or C terminus (Scrib-GFP) with EGFP. ΔCter lacks the region C-terminal to the PDZ domains, which is contained in the Cter construct. LRR and PDZ encode the N-terminal LRR-LAPSD or the C-terminal four PDZ domains, respectively. 4PDZ contains the PDZ domains only. aa, amino acids. (D) Scrib associates with IFs via the region containing the four PDZ domains. EGFP-hScrib WT, LRR, and PDZ were immunoprecipitated from sparse MDCK cells and associated vimentin and keratin 18 as detected by Western blot analysis (a). The detection of ZO-2 served as a positive control for the interaction with hScrib WT or PDZ and as a negative control for the interaction with LRR (Métaiset al., 2005). An aliquot (5%) of the cell lysate was directly subjected to Western blot analysis to monitor the expression levels of vimentin, keratin 18, and ZO-2 (Input) (b). Scrib directly binds to vimentin via its PDZ domains. Purified in vitro-polymerized (c) or nonpolymerized (d) vimentin and in vitro-translated HA-mScrib LRR or HA-mScrib PDZ were combined and incubated. Vimentin was then immunoprecipitated and vimentin or HA-mScrib detected by Western blot. Normal IgG served as a negative control. (E) hScrib PDZ localizes to vimentin filaments in sparse MDCK cells. The different hScrib constructs tagged with EGFP were expressed in MDCK cells and visualized by Western blot analysis (a) or fluorescence microscopy (b–o). Note that both N- and C-terminally tagged Scrib (b and c) and only constructs containing the PDZ domains (d–f, j–l, and m–o) show extensive filamentous localization.
siRNA-mediated depletion of endogenous vimentin and Scrib in MDCK cells. (A) Silencing of Scrib and vimentin monitored by immunofluorescence microscopy. Scrib (a–d, red) and vimentin (e–h, white) were visualized in MDCK cells treated for 3 d with a nontargeting siRNA (a and e) or siRNAs to vimentin (b and f), Scrib (c and g), or both Scrib and vimentin (d and h). (B) Silencing of Scrib and vimentin monitored by Western blot analysis. Scrib and vimentin protein levels in lysates of cells treated with siRNA over a 6-d period were monitored by Western blot on days 2, 4, and 6. Keratin 18 was detected to monitor for equal cell lysate loading.
Silencing of Scrib or vimentin expression in MDCK cells leads to defects in cell morphology and Golgi complex orientation during directed cell migration. (A) Aberrant morphology. Monolayers of cells treated with nontargeting (a, e, and i), vimentin (b, f, and j), Scrib (c, g, and k), or Scrib and vimentin (d, h, and l) siRNA were wounded and stained with an antibody to ZO-2 (a–d) to visualize the cell outline. Scrib (e–h) and vimentin (i–l) were stained to monitor the effectiveness of the siRNA treatment. Note how in control cells the long axis of the cells is directed toward the wound edge (bottom of the images), whereas it is random in cells treated with the specific siRNAs. (B) Monolayers of cells treated with nontargeting (a), vimentin (b), Scrib (c), or Scrib and vimentin (d) siRNA were wounded and stained with an antibody to thecis-Golgi marker GM130 (red) and DAPI (blue) to label nuclei. The wound edge is demarcated with a white line. (e) Golgi complex orientation relative to the nucleus and the migration front was quantified as described inMaterials and Methods. Shown is the fraction of leading edge cellswith correctly polarized Golgi complexes that position in front of the nucleus, facing the wound. Results represent the means of three independent experiments, in which at least 400 cells where scored for each condition. Error bars, SD of the mean. A red line indicates basal levels for a random orientation of 33%. (f) Schematic representation of Golgi complex orientation. The position of Golgi complex relative to the nucleus (blue) and wound edge was determined for ∼30 individual cells for each siRNA treatment and plotted. The shaded sector from 30° to 150° faces the wound edge and is bisected perpendicular to this edge. Note how the positioning of the Golgi complex of most control siRNA-treated cells falls within this sector, whereas that of cells where vimentin, Scrib, or both had been silenced is randomized.
Slower wound closure rates due to a less directed migration of MDCK cells treated with Scrib or vimentin siRNA. (A) Wound closure. Monolayers of cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were wounded and allowed to migrate for 16 h. Images were taken after wounding (0 h; s a–d) or 16 h of migration (e–h). The black marks at the bottom of the dishes allow alignment of the wounds. Panels shown are representative of at least three independent experiments. (B) Quantification of cell migration directionality using live cell tracking. The X-Y graphs represent migration coordinates of 10 different cells at the wound edge treated with nontargeting (a), vimentin (b), Scrib (c), or vimentin and Scrib (d) siRNA, tracked over time 4 d after siRNA transfection. Start points for the different cells were adjusted to (0,0) coordinates. Results are representative of at least three independent experiments. (e) Tortuosity was scored for at least 30 individual cells for each siRNA treatment (n = 3; p< 0.01–0.001, Student'st test). A value of 1 indicates linear movement. Error bars represent SD.
Silencing of Scrib and vimentin expression affects cell–cell aggregation and spreading. (A) Cell aggregation. MDCK cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were allowed to aggregate in a hanging drop and photographed (a–d). (B) Cell spreading. Cell aggregates were transferred from the hanging drop onto coverslips and allowed to adhere and spread (e–h). Note how cells treated with specific siRNAs form less compact aggregates (b–d) and show enhanced spreading (f–h) compared with control cells (a and e, respectively). Assays were carried out 4 d after siRNA transfection.
Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin. (A) Vimentin expression in MDCK cells was silenced using siRNA over 3 d. Cells were subsequently reseeded to sparse and confluent cultures, and Scrib protein levels were monitored by Western blot analysis on day 4. K18 was detected to check for equal cell lysate loading. (B) Quantitative representation of Scrib down-regulation relative to levels of vimentin silencing in MDCK cells. (C) MDCK cells expressing EGFP-hScrib WT (∼250 kDa), LRR (∼130 kDa), PDZ (∼150 kDa), or, as a negative control, EGFP alone, were treated with vimentin (+) or nontargeting (−) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input. (D) MDCK cells expressing EGFP-hScrib WT (a–d), LRR (e–h), or PDZ (i–l) were treated with nontargeting (a, b, e, f, I, and j) or vimentin (c, d, g, h, k, and l) siRNA and EGFP-hScrib (b, f, j, d, h, and l; green) and vimentin (a, e, i, c, g, and k; red) expression was visualized by fluorescence microscopy. (E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18, or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F–H) Effect of a proteasome inhibitor on Scrib turnover. (F) Western blot. MDCK cells expressing EGFP-hScrib WT were treated with vimentin (+) or nontargeting (−) siRNA for 3 d and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (also see H). Actin served as a control for equal lysate input. (G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 h (a and b) or 9 h (c and d) and EGFP-hScrib (a and c; green) and vimentin (b and d; red) expression was visualized by fluorescence microscopy. (H) Western blot for endogenous Scrib. MDCK cells were treated with vimentin (+) or nontargeting (−) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input. (I) hScrib-EGFP of nontargeting or vimentin siRNA-treated MDCK cells in the 9 h presence (+) or absence (−) of proteasome inhibitor was immunoprecipitated and ubiquitinylated hScrib detected by Western blot. Normal IgG served as a negative control.
Comment in
- Mol Biol Cell. 20:2809.
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