Figure 1.
Interaction of the RPEL repeats of scapinin with cellular actin.
(A) The primary structure of scapinin. The N-terminal region that is conserved in the scapinin/phactr family (NC), the proline-rich region (PR), the three tandem repeats of PREL motifs (RPEL repeats 1, 2, and 3), and the PP1-binding domains (PP1) are indicated. In addition to the N-terminal conserved region (NC), the RPEL-repeat and PP1-binding domains are highly conserved in the scapinin/phactr family. (B) Three RPEL motifs are compared. The RPEL motif (pfam PF02755) is named according to the conserved amino acid sequence, RPxxxEL. (C) Interactions between the RPEL repeats of scapinin and cellular actin. The RPEL repeats of scapinin were produced as fusion proteins with glutathione S-transferase (GST) in E. Coli as illustrated. In the RP>AA mutant, both the arginine and proline residues in the second motif of the RPEL repeats were substituted with alanine residues. Purified GST-RPEL constructs were separated by SDS-polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). GST-RPEL constructs were covalently conjugated to CNBr-agarose beads (5 mg protein/ml bed volume) and subjected to a pull-down assay with HL-60 cell lysates (see ‘Methods’). Actin in the pull-down-samples was determined by Western blotting (WB) with anti-actin antibody. The GST-RPEL construct (350–465 aa) contained substantial amounts of cleaved products (see ‘Results’).
Figure 2.
The RPEL repeats of scapinin interact with purified skeletal muscle actin and inhibit actin polymerization in vitro.
(A) GST or GST-RPEL repeats (350–422 aa) were covalently conjugated to CNBr-agarose beads (as in Figure 1C) and were incubated with purified skeletal muscle actin. After washing with RIPA buffer (0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 mM Tris-HCl pH 8.0, 150 mM NaCl), bound proteins were eluted with SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, and stained with coomassie brilliant blue (CBB). GST and GST-RPEL proteins (shown by a asterisk) were partly released from the beads by elution with SDS sample buffer. (B) Inhibition of actin polymerization by the RPEL repeats. Skeletal muscle actin (18 µM) was incubated with GST- RPEL repeats (350–422 aa) or GST at the indicated concentrations to polymerize at room temperature for 30 minutes, and then filamentous actin (P) and monomeric actin (S) were separated by ultracentrifugation. Aliquots were analyzed by SDS polyacrylamide gel electrophoresis and stained with coomassie brilliant blue (CBB). Since the contamination of supernatants in the pellet fraction was technically inevitable, a small portion of GST-RPEL construct was seen in the pellet. (C) The density of each actin band was measured by a densitometer and plotted.
Figure 3.
Induction of scapinin expression in Hela cells and its effect on cell proliferation.
(A) Tetracycline-induced expression of scapinin in Hela cells. Hela cells were cultured with tetracycline at the indicated concentrations for 24 hours. Cell lysates were separated by SDD-polyacrylamide gel electrophoresis, and scapinin expression levels were measured with Western blotting using anti-scapinin monoclonal antibody. Human whole brain lysate (Clontech Laboratories Inc.) was also loaded. Twenty microgram proteins were applied to each lane. Scapinin expression levels of Hela cells were expressed as folds against the whole brain lysate. (C) The effect of scapinin on the proliferation of Hela cells. Hela cells were plated at 1×104 cells/ well in 96-well plates and cultured with (open bars) or without (closed bars) 0.1 µg/ml tetracycline. At each time point, the number of viable cells was assessed by an MTT assay, and the absorbance was measured at a wavelength of 570 nm with a 96-well plate reader. Data are expressed as mean±SEM of four separate experiments.
Figure 4.
Enhancement of cell spreading and motility by scapinin.
(A) Enhancement of cell spreading by scapinin. Inducible Hela cells were seeded onto an 8-chambered glass-slide and then cultured with (Tet+) or without (Tet−) 0.1 µg/ml tetracycline. Cell morphology was monitored with a light microscope and photographed at 12 hours. (B) Inducible Hela cells were treated as in (A), and cell morphology was monitored at specified times. Adherent and non-adherent cells were counted under a microscope. Data are expressed as mean±SED from four independent experiments. Student's t test: *: P<0.05; **: P<0.01. The parental Hela cells were also cultured with (Tet+) or without (Tet−) of tetracycline, and cell morphology was monitored. (C) Enhancement of cell motility by scapinin. To measure cell motility, we used a wound healing assay. Hela cells were cultured on 6-well plates until confluence. The confluent monolayer cultures were treated with (Tet+) or without (Tet−) 0.1 µg/ml tetracycline for 4 hours and were then wounded with a straight scratch using a yellow pipette tip. After washing them three times with serum-free DMEM, the wounded monolayer cultures were further incubated (Tet+) with or without (Tet−) 0.1 µg/ml tetracycline in DMEM containing 1% fetal bovine serum. To reduce cell growth, the serum concentration was reduced to 1%. At 1, 24, and 48 hours after the wounding, the cell monolayer was photographed. The front of cell migration at 24 hours was shown in (D).
Figure 5.
Distribution of scapinin and actin in Hela cells.
(A) Hela cells grown on glass coverslips were cultured in the presence (Tet+) or absence (Tet−) of 0.1 µg/ml tetracycline and were then fixed at 20 hours. After permeabilization, the distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively. Scapinin and actin are colocalized (arrows). (B) Confocal microscopic observation. Hela cells were cultured in the presence of 0.1 µg/ml tetracycline for 20 hours and were then stained with anti-scapinin antibody (green) and rhodamine-phalloidin (red) as in (A). Scapinin and actin are colocalized (arrows). Bar: 20 µm. (C) Absence of scapinin in actin stress fibers. Hela cells grown on a glass coverslip were cultured in the presence of 0.1 µg/ml tetracycline and were then fixed at 8 hours. The distribution of scapinin and the actin cytoskeleton were visualized by staining with anti-scapinin antibody (green) and rhodamine-phalloidin (red), respectively as (A). There are four cells; two cells express low levels of scapinin (asterisks), and the other two cells express high levels of scapinin (arrow heads). Bar: 20 µm.
Figure 6.
GFP-scapinin-induced morphological changes in Cos7 cells.
pEGFP empty vectors (A) and pEGFP-scapinin (B) were transfected into Cos7 cells, and the cells were cultured for 16 hours. The cell morphology and the distribution of GFP-scapinin were monitored with fluorescent microscopy and photographed. (C) The distribution of GFP-scapinin in ruffles. (D) Colocalization of scapinin and actin in ruffles. Cos7 cells expressing GFP-scapinin were fixed at 16 hours and then stained with rhodamine-phalloidin (red) to visualize actin. Localization of GFP-scapinin (green) and actin (red) was observed with a confocal microscopy. Ruffles were shown by arrows. Bars: 20 µm.
Figure 7.
The roles of the RPEL-repeat and PP1-binding domains in scapinin-cell spreading.
(A) The pEGFP-scapinin mutants used in this study are illustrated. (B) Each construct shown in (A) was transfected into Cos7 cells. The cells were cultured for 24 hours and lysed in 0.5% Triton X-100/cytoskeleton buffer at 24 hours. GFP-scapinins were immunoprecipitated with anti-GFP antibody, and immunocomplexes were collected with protein A-agarose beads, washed, and subjected to Western blotting with anti-scapinin monoclonal antibody, anti-actin monoclonal antibody, and anti-PP1 polyclonal antibodies, respectively. (C) Each pEGFP-scapinin mutant was transfected into Cos7 cells. Cells were cultured for 16 hours and then monitored under a fluorescent microscope and photographed. Bar: 20 µm.
Figure 8.
Time course of morphological changes of Cos7 cells induced by a PP1-binding deficient mutant of GFP-scapinin (F515A).
After transfection of pEGFP-scapinin F515A mutant, morphological changes of Cos7 cells were monitored and photographed at indicated times under a fluorescence microscopy. Bar: 20 µm.