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Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Sagara J, Arata T, Taniguchi S - PLoS ONE (2009)

Bottom Line: PP1-binding deficient mutants strongly induced cell retraction.Long and branched cytoplasmic processes were developed during the cell retraction.These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan. sagaraj@shinshu-u.ac.jp

ABSTRACT
Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

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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).
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pone-0004247-g004: 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).

Mentions: Cell spreading was significantly enhanced by expression of scapinin (Fig. 4A). Scapinin-expressing cells adhered to glassslides faster than non-expressing cells (Fig. 4B). Next we measured cell area of adherent cells at 20 hours. To focus on adherent cells, rounded cells were excluded from this measurement. Cell area of adherent cells of non-expressing (Tet−) and scapinin-expressing (Tet+) cells were 586±53 µm2 and 883±95 µm2 (mean±SD, n = 50), respectively. Cell spreading of parental Hela cells was not enhanced by tetracycline treatment (Fig. 4B). Notably, scapinin-expressing cells frequently exhibited elongated shapes that were rarely seen in the parental Hela cells used in this study.


Scapinin, the protein phosphatase 1 binding protein, enhances cell spreading and motility by interacting with the actin cytoskeleton.

Sagara J, Arata T, Taniguchi S - PLoS ONE (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2626280&req=5

pone-0004247-g004: 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).
Mentions: Cell spreading was significantly enhanced by expression of scapinin (Fig. 4A). Scapinin-expressing cells adhered to glassslides faster than non-expressing cells (Fig. 4B). Next we measured cell area of adherent cells at 20 hours. To focus on adherent cells, rounded cells were excluded from this measurement. Cell area of adherent cells of non-expressing (Tet−) and scapinin-expressing (Tet+) cells were 586±53 µm2 and 883±95 µm2 (mean±SD, n = 50), respectively. Cell spreading of parental Hela cells was not enhanced by tetracycline treatment (Fig. 4B). Notably, scapinin-expressing cells frequently exhibited elongated shapes that were rarely seen in the parental Hela cells used in this study.

Bottom Line: PP1-binding deficient mutants strongly induced cell retraction.Long and branched cytoplasmic processes were developed during the cell retraction.These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Nagano, Japan. sagaraj@shinshu-u.ac.jp

ABSTRACT
Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.

Show MeSH
Related in: MedlinePlus