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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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Dynamics of SH3YL1, SHIP2, and PI(3,4)P2 at the circular dorsal ruffle. (A) Time-lapse images of F-actin probed by Lifeact-mCherry transfected in NIH3T3 cells stimulated with 20 ng/ml PDGF. Arrows indicate precursor structures. Bar, 10 µm. (B and C) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-2×Tapp1PH (B) or HA-SH3YL1 and Myc-SHIP2 (C) were stimulated with 20 ng/ml PDGF for 3 and 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bars, 10 µm. Insets show the boxed areas at high magnification. (D) A model for circular dorsal ruffle formation by SH3YL1 and SHIP2. See Discussion for details.
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fig8: Dynamics of SH3YL1, SHIP2, and PI(3,4)P2 at the circular dorsal ruffle. (A) Time-lapse images of F-actin probed by Lifeact-mCherry transfected in NIH3T3 cells stimulated with 20 ng/ml PDGF. Arrows indicate precursor structures. Bar, 10 µm. (B and C) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-2×Tapp1PH (B) or HA-SH3YL1 and Myc-SHIP2 (C) were stimulated with 20 ng/ml PDGF for 3 and 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bars, 10 µm. Insets show the boxed areas at high magnification. (D) A model for circular dorsal ruffle formation by SH3YL1 and SHIP2. See Discussion for details.

Mentions: To examine the molecular dynamics of circular dorsal ruffle formation, the distribution of F-actin, PI(3,4)P2, SH3YL1, and SHIP2 was monitored in PDGF-stimulated NIH3T3 cells that were transfected with Lifeact-mCherry, a probe for F-actin (Riedl et al., 2008), by time-lapse microscopy. Consistent with the data shown in Fig. 3 A, numerous short F-actin filaments, which appeared to be dorsal ruffle precursors, were observed 3–4 min after stimulation followed by the formation of an F-actin–enriched circular structure at 6 min (Fig. 8 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)

Dynamics of SH3YL1, SHIP2, and PI(3,4)P2 at the circular dorsal ruffle. (A) Time-lapse images of F-actin probed by Lifeact-mCherry transfected in NIH3T3 cells stimulated with 20 ng/ml PDGF. Arrows indicate precursor structures. Bar, 10 µm. (B and C) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-2×Tapp1PH (B) or HA-SH3YL1 and Myc-SHIP2 (C) were stimulated with 20 ng/ml PDGF for 3 and 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bars, 10 µm. Insets show the boxed areas at high magnification. (D) A model for circular dorsal ruffle formation by SH3YL1 and SHIP2. See Discussion for details.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Dynamics of SH3YL1, SHIP2, and PI(3,4)P2 at the circular dorsal ruffle. (A) Time-lapse images of F-actin probed by Lifeact-mCherry transfected in NIH3T3 cells stimulated with 20 ng/ml PDGF. Arrows indicate precursor structures. Bar, 10 µm. (B and C) NIH3T3 cells transiently transfected with HA-SH3YL1 and Myc-2×Tapp1PH (B) or HA-SH3YL1 and Myc-SHIP2 (C) were stimulated with 20 ng/ml PDGF for 3 and 5 min, fixed, and stained with anti-HA, anti-Myc antibodies, and Alexa Fluor 647–phalloidin. Bars, 10 µm. Insets show the boxed areas at high magnification. (D) A model for circular dorsal ruffle formation by SH3YL1 and SHIP2. See Discussion for details.
Mentions: To examine the molecular dynamics of circular dorsal ruffle formation, the distribution of F-actin, PI(3,4)P2, SH3YL1, and SHIP2 was monitored in PDGF-stimulated NIH3T3 cells that were transfected with Lifeact-mCherry, a probe for F-actin (Riedl et al., 2008), by time-lapse microscopy. Consistent with the data shown in Fig. 3 A, numerous short F-actin filaments, which appeared to be dorsal ruffle precursors, were observed 3–4 min after stimulation followed by the formation of an F-actin–enriched circular structure at 6 min (Fig. 8 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