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EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.

Maul RS, Song Y, Amann KJ, Gerbin SC, Pollard TD, Chang DD - J. Cell Biol. (2003)

Bottom Line: EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex.Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex.We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.

ABSTRACT
Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

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EPLIN cross-links and bundles F-actin. (A) Low speed pelleting assay. Samples containing 10 μM F-actin and 0.4–2.5 μM his-EPLIN-α or 0.4– 2.5 μM α-actinin were centrifuged at 8,000 g for 20 min. The negative control, BSA, was used at 2.5 μM. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of his-EPLIN-α, α-actinin, and BSA are indicated by filled circles. (B and C) Electron microscopy. 2 μM actin was polymerized with 1 μM his-EPLIN-α (B) or 1 μM BSA (C), negatively stained with uranyl acetate and visualized by electron microscopy. The boxed areas were enlarged (insets) to better illustrate actin filaments.
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fig4: EPLIN cross-links and bundles F-actin. (A) Low speed pelleting assay. Samples containing 10 μM F-actin and 0.4–2.5 μM his-EPLIN-α or 0.4– 2.5 μM α-actinin were centrifuged at 8,000 g for 20 min. The negative control, BSA, was used at 2.5 μM. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of his-EPLIN-α, α-actinin, and BSA are indicated by filled circles. (B and C) Electron microscopy. 2 μM actin was polymerized with 1 μM his-EPLIN-α (B) or 1 μM BSA (C), negatively stained with uranyl acetate and visualized by electron microscopy. The boxed areas were enlarged (insets) to better illustrate actin filaments.

Mentions: The increase in actin stress fibers observed in EPLIN-overexpressing cells, together with its localization to the actin cytoskeleton, raised the possibility that it may bundle actin filaments. Bundled or cross-linked actin filaments can be detected by sedimentation at low speed where free actin filaments remain in the supernatant. Because GST can form a homodimer (Ji et al., 1992), bundling assays were performed with his-EPLIN-α. His-EPLIN-α caused the majority of actin filaments to pellet during low speed centrifugation (Fig. 4 A). At the same molar concentration, EPLIN-α pelleted actin filaments more efficiently than α-actinin, a dimeric cross-linking protein with two actin-binding sites. A control protein, BSA, remained in the supernatant with the actin filaments. We used electron microscopy of negatively stained specimens to confirm that bundling was responsible for actin filaments pelleting with EPLIN during low speed centrifugation. Actin filaments formed thick, tightly packed bundles in the presence of EPLIN-α (Fig. 4 B). In the presence of BSA (used as a negative control), the F-actin remained as thin wavy filaments (Fig. 4 C).


EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.

Maul RS, Song Y, Amann KJ, Gerbin SC, Pollard TD, Chang DD - J. Cell Biol. (2003)

EPLIN cross-links and bundles F-actin. (A) Low speed pelleting assay. Samples containing 10 μM F-actin and 0.4–2.5 μM his-EPLIN-α or 0.4– 2.5 μM α-actinin were centrifuged at 8,000 g for 20 min. The negative control, BSA, was used at 2.5 μM. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of his-EPLIN-α, α-actinin, and BSA are indicated by filled circles. (B and C) Electron microscopy. 2 μM actin was polymerized with 1 μM his-EPLIN-α (B) or 1 μM BSA (C), negatively stained with uranyl acetate and visualized by electron microscopy. The boxed areas were enlarged (insets) to better illustrate actin filaments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172667&req=5

fig4: EPLIN cross-links and bundles F-actin. (A) Low speed pelleting assay. Samples containing 10 μM F-actin and 0.4–2.5 μM his-EPLIN-α or 0.4– 2.5 μM α-actinin were centrifuged at 8,000 g for 20 min. The negative control, BSA, was used at 2.5 μM. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of his-EPLIN-α, α-actinin, and BSA are indicated by filled circles. (B and C) Electron microscopy. 2 μM actin was polymerized with 1 μM his-EPLIN-α (B) or 1 μM BSA (C), negatively stained with uranyl acetate and visualized by electron microscopy. The boxed areas were enlarged (insets) to better illustrate actin filaments.
Mentions: The increase in actin stress fibers observed in EPLIN-overexpressing cells, together with its localization to the actin cytoskeleton, raised the possibility that it may bundle actin filaments. Bundled or cross-linked actin filaments can be detected by sedimentation at low speed where free actin filaments remain in the supernatant. Because GST can form a homodimer (Ji et al., 1992), bundling assays were performed with his-EPLIN-α. His-EPLIN-α caused the majority of actin filaments to pellet during low speed centrifugation (Fig. 4 A). At the same molar concentration, EPLIN-α pelleted actin filaments more efficiently than α-actinin, a dimeric cross-linking protein with two actin-binding sites. A control protein, BSA, remained in the supernatant with the actin filaments. We used electron microscopy of negatively stained specimens to confirm that bundling was responsible for actin filaments pelleting with EPLIN during low speed centrifugation. Actin filaments formed thick, tightly packed bundles in the presence of EPLIN-α (Fig. 4 B). In the presence of BSA (used as a negative control), the F-actin remained as thin wavy filaments (Fig. 4 C).

Bottom Line: EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex.Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex.We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.

ABSTRACT
Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

Show MeSH
Related in: MedlinePlus