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SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain.

Hasegawa J, Tokuda E, Tenno T, Tsujita K, Sawai H, Hiroaki H, Takenawa T, Itoh T - J. Cell Biol. (2011)

Bottom Line: Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation.Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure.These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Membrane Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.

ABSTRACT
Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

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SH3YL1 is necessary for dorsal ruffle formation. (A) Anti-SH3YL1 immunoblots of NIH3T3 cells transfected with control or SH3YL1-specific siRNA. (B) Phalloidin staining of control or SH3YL1-depleted NIH3T3 cells treated with PDGF for 5 min. Bar, 10 µm. (C) Percentages of cells generating at least one circular dorsal ruffle. Rescue experiments were performed by transfecting HA-SH3YL1 constructs (blot, expression levels of the indicated constructs) to recover circular dorsal ruffle formation. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01; *, P < 0.05. (D) Control or SH3YL1-depleted NIH3T3 cells were allowed to internalize the fluid-phase marker rhodamine-dextran (0.2 mg/ml; shown in red) for 10 min and then stained with Hoechst (cyan). Bar, 10 µm. (E) NIH3T3 cells transfected with indicated siRNAs were subjected to HRP uptake assay for 10 min with or without PDGF stimulation (20 ng/ml). Results are a mean (SD) of three independent experiments.
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fig4: SH3YL1 is necessary for dorsal ruffle formation. (A) Anti-SH3YL1 immunoblots of NIH3T3 cells transfected with control or SH3YL1-specific siRNA. (B) Phalloidin staining of control or SH3YL1-depleted NIH3T3 cells treated with PDGF for 5 min. Bar, 10 µm. (C) Percentages of cells generating at least one circular dorsal ruffle. Rescue experiments were performed by transfecting HA-SH3YL1 constructs (blot, expression levels of the indicated constructs) to recover circular dorsal ruffle formation. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01; *, P < 0.05. (D) Control or SH3YL1-depleted NIH3T3 cells were allowed to internalize the fluid-phase marker rhodamine-dextran (0.2 mg/ml; shown in red) for 10 min and then stained with Hoechst (cyan). Bar, 10 µm. (E) NIH3T3 cells transfected with indicated siRNAs were subjected to HRP uptake assay for 10 min with or without PDGF stimulation (20 ng/ml). Results are a mean (SD) of three independent experiments.

Mentions: Because SH3YL1 localized to the dorsal ruffles, we assessed whether it was necessary for the formation of dorsal ruffles. Transfection of SH3YL1-targeting siRNAs resulted in the depletion of >90% of SH3YL1 protein but did not alter actin levels (Fig. 4 A). Dorsal ruffles were formed in ∼60% of control siRNA–treated cells after PDGF stimulation, whereas significantly fewer ruffles were formed in SH3YL1 siRNA–treated cells (Fig. 4, B and C). This suppression was alleviated by the exogenous wild-type SH3YL1 but not by ΔSH3, M1, or M2 constructs (Fig. 4 C), suggesting that the presence of both the SH3 domain and a functional SYLF domain is necessary.


SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain.

Hasegawa J, Tokuda E, Tenno T, Tsujita K, Sawai H, Hiroaki H, Takenawa T, Itoh T - J. Cell Biol. (2011)

SH3YL1 is necessary for dorsal ruffle formation. (A) Anti-SH3YL1 immunoblots of NIH3T3 cells transfected with control or SH3YL1-specific siRNA. (B) Phalloidin staining of control or SH3YL1-depleted NIH3T3 cells treated with PDGF for 5 min. Bar, 10 µm. (C) Percentages of cells generating at least one circular dorsal ruffle. Rescue experiments were performed by transfecting HA-SH3YL1 constructs (blot, expression levels of the indicated constructs) to recover circular dorsal ruffle formation. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01; *, P < 0.05. (D) Control or SH3YL1-depleted NIH3T3 cells were allowed to internalize the fluid-phase marker rhodamine-dextran (0.2 mg/ml; shown in red) for 10 min and then stained with Hoechst (cyan). Bar, 10 µm. (E) NIH3T3 cells transfected with indicated siRNAs were subjected to HRP uptake assay for 10 min with or without PDGF stimulation (20 ng/ml). Results are a mean (SD) of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105542&req=5

fig4: SH3YL1 is necessary for dorsal ruffle formation. (A) Anti-SH3YL1 immunoblots of NIH3T3 cells transfected with control or SH3YL1-specific siRNA. (B) Phalloidin staining of control or SH3YL1-depleted NIH3T3 cells treated with PDGF for 5 min. Bar, 10 µm. (C) Percentages of cells generating at least one circular dorsal ruffle. Rescue experiments were performed by transfecting HA-SH3YL1 constructs (blot, expression levels of the indicated constructs) to recover circular dorsal ruffle formation. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01; *, P < 0.05. (D) Control or SH3YL1-depleted NIH3T3 cells were allowed to internalize the fluid-phase marker rhodamine-dextran (0.2 mg/ml; shown in red) for 10 min and then stained with Hoechst (cyan). Bar, 10 µm. (E) NIH3T3 cells transfected with indicated siRNAs were subjected to HRP uptake assay for 10 min with or without PDGF stimulation (20 ng/ml). Results are a mean (SD) of three independent experiments.
Mentions: Because SH3YL1 localized to the dorsal ruffles, we assessed whether it was necessary for the formation of dorsal ruffles. Transfection of SH3YL1-targeting siRNAs resulted in the depletion of >90% of SH3YL1 protein but did not alter actin levels (Fig. 4 A). Dorsal ruffles were formed in ∼60% of control siRNA–treated cells after PDGF stimulation, whereas significantly fewer ruffles were formed in SH3YL1 siRNA–treated cells (Fig. 4, B and C). This suppression was alleviated by the exogenous wild-type SH3YL1 but not by ΔSH3, M1, or M2 constructs (Fig. 4 C), suggesting that the presence of both the SH3 domain and a functional SYLF domain is necessary.

Bottom Line: Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation.Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure.These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Membrane Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.

ABSTRACT
Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.

Show MeSH