Limits...
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.

Show MeSH

Related in: MedlinePlus

SHIP2 is required for circular dorsal ruffle formation. (A) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-SHIP2 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bar, 10 µm. (B) Anti-SHIP2 immunoblots of NIH3T3 cells transfected with control or SHIP2-specific siRNA. (C) PDGF-induced circular dorsal ruffle formation was reduced in SHIP2-depleted NIH3T3 cells. Phalloidin staining of cells treated with control or SHIP2 siRNAs is shown. The arrow indicates peripheral ruffles, and arrowheads indicate straight dorsal ruffles. Bar, 10 µm. (D) Percentages of cells generating at least one circular dorsal ruffle, peripheral ruffle, or straight dorsal ruffle. Rescue experiments were performed by transfecting Myc-SHIP2 constructs (blot, expression levels of the indicated constructs) to recover each ruffle formation. WT, wild type. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01 and *, P < 0.05. (E) SHIP2-depleted NIH3T3 cells transiently transfected with HA-SH3YL1 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA antibodies and rhodamine-phalloidin. Arrows indicate straight dorsal ruffles. Bar, 10 µm. (F) Rhodamine-dextran (red) and Hoechst (cyan) images of NIH3T3 cells treated with SHIP2 or control siRNA and incubated for 10 min with 0.2 mg/ml rhodamine-dextran. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3105542&req=5

fig6: SHIP2 is required for circular dorsal ruffle formation. (A) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-SHIP2 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bar, 10 µm. (B) Anti-SHIP2 immunoblots of NIH3T3 cells transfected with control or SHIP2-specific siRNA. (C) PDGF-induced circular dorsal ruffle formation was reduced in SHIP2-depleted NIH3T3 cells. Phalloidin staining of cells treated with control or SHIP2 siRNAs is shown. The arrow indicates peripheral ruffles, and arrowheads indicate straight dorsal ruffles. Bar, 10 µm. (D) Percentages of cells generating at least one circular dorsal ruffle, peripheral ruffle, or straight dorsal ruffle. Rescue experiments were performed by transfecting Myc-SHIP2 constructs (blot, expression levels of the indicated constructs) to recover each ruffle formation. WT, wild type. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01 and *, P < 0.05. (E) SHIP2-depleted NIH3T3 cells transiently transfected with HA-SH3YL1 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA antibodies and rhodamine-phalloidin. Arrows indicate straight dorsal ruffles. Bar, 10 µm. (F) Rhodamine-dextran (red) and Hoechst (cyan) images of NIH3T3 cells treated with SHIP2 or control siRNA and incubated for 10 min with 0.2 mg/ml rhodamine-dextran. Bar, 10 µm.

Mentions: To determine the function of SHIP2 in dorsal ruffle formation, NIH3T3 cells were transfected with Myc-SHIP2 and HA-SH3YL1 and stimulated with PDGF. As a result, significant colocalization of SH3YL1 and SHIP2 was observed at the circular dorsal ruffles (Fig. 6 A).


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)

SHIP2 is required for circular dorsal ruffle formation. (A) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-SHIP2 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bar, 10 µm. (B) Anti-SHIP2 immunoblots of NIH3T3 cells transfected with control or SHIP2-specific siRNA. (C) PDGF-induced circular dorsal ruffle formation was reduced in SHIP2-depleted NIH3T3 cells. Phalloidin staining of cells treated with control or SHIP2 siRNAs is shown. The arrow indicates peripheral ruffles, and arrowheads indicate straight dorsal ruffles. Bar, 10 µm. (D) Percentages of cells generating at least one circular dorsal ruffle, peripheral ruffle, or straight dorsal ruffle. Rescue experiments were performed by transfecting Myc-SHIP2 constructs (blot, expression levels of the indicated constructs) to recover each ruffle formation. WT, wild type. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01 and *, P < 0.05. (E) SHIP2-depleted NIH3T3 cells transiently transfected with HA-SH3YL1 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA antibodies and rhodamine-phalloidin. Arrows indicate straight dorsal ruffles. Bar, 10 µm. (F) Rhodamine-dextran (red) and Hoechst (cyan) images of NIH3T3 cells treated with SHIP2 or control siRNA and incubated for 10 min with 0.2 mg/ml rhodamine-dextran. Bar, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105542&req=5

fig6: SHIP2 is required for circular dorsal ruffle formation. (A) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-SHIP2 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bar, 10 µm. (B) Anti-SHIP2 immunoblots of NIH3T3 cells transfected with control or SHIP2-specific siRNA. (C) PDGF-induced circular dorsal ruffle formation was reduced in SHIP2-depleted NIH3T3 cells. Phalloidin staining of cells treated with control or SHIP2 siRNAs is shown. The arrow indicates peripheral ruffles, and arrowheads indicate straight dorsal ruffles. Bar, 10 µm. (D) Percentages of cells generating at least one circular dorsal ruffle, peripheral ruffle, or straight dorsal ruffle. Rescue experiments were performed by transfecting Myc-SHIP2 constructs (blot, expression levels of the indicated constructs) to recover each ruffle formation. WT, wild type. Results are a mean (SD) of three independent experiments; 200 cells counted (100 cells for the rescue experiments) per experiment. **, P < 0.01 and *, P < 0.05. (E) SHIP2-depleted NIH3T3 cells transiently transfected with HA-SH3YL1 were serum starved, stimulated with 20 ng/ml PDGF for 5 min, fixed, and stained with anti-HA antibodies and rhodamine-phalloidin. Arrows indicate straight dorsal ruffles. Bar, 10 µm. (F) Rhodamine-dextran (red) and Hoechst (cyan) images of NIH3T3 cells treated with SHIP2 or control siRNA and incubated for 10 min with 0.2 mg/ml rhodamine-dextran. Bar, 10 µm.
Mentions: To determine the function of SHIP2 in dorsal ruffle formation, NIH3T3 cells were transfected with Myc-SHIP2 and HA-SH3YL1 and stimulated with PDGF. As a result, significant colocalization of SH3YL1 and SHIP2 was observed at the circular dorsal ruffles (Fig. 6 A).

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
Related in: MedlinePlus