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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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Localization of SH3YL1 at the dorsal ruffle. (A) NIH3T3 cells expressing HA-SH3YL1 (full length) were stimulated with 20 ng/ml PDGF for the indicated times and stained with anti-HA antibodies and rhodamine-phalloidin. Insets, precursor structures at a high magnification. Where indicated, 25 µM LY294002 was applied for 30 min before stimulation. (B) Localization of wild-type (WT) HA-SH3YL1 and mutant (M1) at 5 min after PDGF stimulation. (A and B) Arrows, circular dorsal ruffles. Bars, 10 µm. (C) Fluorescence intensity profiles of white lines in B. (D) Fluorescence intensities in the cytosol were subtracted from those at circular dorsal ruffles. The ratio of anti-HA versus mCherry are presented. Results are a mean (SD) of four experiments.
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fig3: Localization of SH3YL1 at the dorsal ruffle. (A) NIH3T3 cells expressing HA-SH3YL1 (full length) were stimulated with 20 ng/ml PDGF for the indicated times and stained with anti-HA antibodies and rhodamine-phalloidin. Insets, precursor structures at a high magnification. Where indicated, 25 µM LY294002 was applied for 30 min before stimulation. (B) Localization of wild-type (WT) HA-SH3YL1 and mutant (M1) at 5 min after PDGF stimulation. (A and B) Arrows, circular dorsal ruffles. Bars, 10 µm. (C) Fluorescence intensity profiles of white lines in B. (D) Fluorescence intensities in the cytosol were subtracted from those at circular dorsal ruffles. The ratio of anti-HA versus mCherry are presented. Results are a mean (SD) of four experiments.

Mentions: Under serum-starved conditions (0 min), HA-tagged SH3YL1 (HA-SH3YL1) was localized to the cytosol in NIH3T3 cells. 2 min after PDGF stimulation, HA-SH3YL1 was recruited to a plasma membrane structure with short actin filaments that were aligned in a circular pattern (Fig. 3 A). This structure appeared to be an immature form of dorsal ruffles, suggesting that SH3YL1 functions at an early stage of dorsal ruffle formation. At 5 min, HA-SH3YL1 accumulated at mature and circular dorsal ruffles that were rich in dense F-actin structures. Consistent with the observations in previous studies (Araki et al., 1996; Suetsugu et al., 2003), dorsal ruffle formation was blocked by LY294002, a PI3K inhibitor (Fig. 3 A), indicating that D3-phosphorylated phosphoinositides and their binding partners are involved in this process.


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)

Localization of SH3YL1 at the dorsal ruffle. (A) NIH3T3 cells expressing HA-SH3YL1 (full length) were stimulated with 20 ng/ml PDGF for the indicated times and stained with anti-HA antibodies and rhodamine-phalloidin. Insets, precursor structures at a high magnification. Where indicated, 25 µM LY294002 was applied for 30 min before stimulation. (B) Localization of wild-type (WT) HA-SH3YL1 and mutant (M1) at 5 min after PDGF stimulation. (A and B) Arrows, circular dorsal ruffles. Bars, 10 µm. (C) Fluorescence intensity profiles of white lines in B. (D) Fluorescence intensities in the cytosol were subtracted from those at circular dorsal ruffles. The ratio of anti-HA versus mCherry are presented. Results are a mean (SD) of four experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Localization of SH3YL1 at the dorsal ruffle. (A) NIH3T3 cells expressing HA-SH3YL1 (full length) were stimulated with 20 ng/ml PDGF for the indicated times and stained with anti-HA antibodies and rhodamine-phalloidin. Insets, precursor structures at a high magnification. Where indicated, 25 µM LY294002 was applied for 30 min before stimulation. (B) Localization of wild-type (WT) HA-SH3YL1 and mutant (M1) at 5 min after PDGF stimulation. (A and B) Arrows, circular dorsal ruffles. Bars, 10 µm. (C) Fluorescence intensity profiles of white lines in B. (D) Fluorescence intensities in the cytosol were subtracted from those at circular dorsal ruffles. The ratio of anti-HA versus mCherry are presented. Results are a mean (SD) of four experiments.
Mentions: Under serum-starved conditions (0 min), HA-tagged SH3YL1 (HA-SH3YL1) was localized to the cytosol in NIH3T3 cells. 2 min after PDGF stimulation, HA-SH3YL1 was recruited to a plasma membrane structure with short actin filaments that were aligned in a circular pattern (Fig. 3 A). This structure appeared to be an immature form of dorsal ruffles, suggesting that SH3YL1 functions at an early stage of dorsal ruffle formation. At 5 min, HA-SH3YL1 accumulated at mature and circular dorsal ruffles that were rich in dense F-actin structures. Consistent with the observations in previous studies (Araki et al., 1996; Suetsugu et al., 2003), dorsal ruffle formation was blocked by LY294002, a PI3K inhibitor (Fig. 3 A), indicating that D3-phosphorylated phosphoinositides and their binding partners are involved in this process.

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