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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 binds directly to both G- and F-actin. (A) Actin monomer pull-down assay with GST-EPLIN. Glutathione-Sepharose beads carrying 2 μg of GST-EPLIN-α (lane 1), GST-VCA (lane 2), and GST (lane 3) were incubated with 50 nM G-actin in 0.5 ml. G-actin bound to the beads was assayed by immunoblotting with anti-actin antibodies (top). In lanes 4–6, varying amounts of G-actin (2.5–7.5% of the input) were included to estimate the amount of bound G-actin. A duplicate gel was stained with Coomassie blue to estimate the amounts of GST proteins present in each lane (bottom). GST-VCA contains a G-actin–binding verprolin (V) homology domain, a cofilin (C) homology domain, and an acidic segment (A) of bovine N-WASp. (B) Actin monomer pull-down assays were performed with GST-EPLIN-α (lane 1), GST-ΔN (lane 2), GST-αΔC (lane 3), GST-αΔC/LIM (lane 4), and GST-LIM (lane 5). (C) Schematic diagrams of EPLIN truncations used in the actin monomer pull-down assay are shown. (D) Actin filament pelleting assay. Samples containing 16 μM polymerized actin and 2 μM EPLIN-α were centrifuged at high speed. Control proteins (BSA, negative controls; α-actinin, positive control) were also used at 2 μM. The presence (+) or absence (−) of actin in the pelleting assay is denoted. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of control proteins and EPLIN-α, or its truncated derivatives, his-EPLINΔN/LIM and his-EPLINαΔC, are indicated by filled circles. (E) Binding of his-EPLIN-α to actin filament. His-EPLIN-α (0–8 μM) was mixed with 10 μM polymerized actin, followed by ultracentrifugation. Amounts of the free and bound his-EPLIN-α in the supernatant and pellet fractions were determined from a digitized Coomassie-stained gel.
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fig3: EPLIN binds directly to both G- and F-actin. (A) Actin monomer pull-down assay with GST-EPLIN. Glutathione-Sepharose beads carrying 2 μg of GST-EPLIN-α (lane 1), GST-VCA (lane 2), and GST (lane 3) were incubated with 50 nM G-actin in 0.5 ml. G-actin bound to the beads was assayed by immunoblotting with anti-actin antibodies (top). In lanes 4–6, varying amounts of G-actin (2.5–7.5% of the input) were included to estimate the amount of bound G-actin. A duplicate gel was stained with Coomassie blue to estimate the amounts of GST proteins present in each lane (bottom). GST-VCA contains a G-actin–binding verprolin (V) homology domain, a cofilin (C) homology domain, and an acidic segment (A) of bovine N-WASp. (B) Actin monomer pull-down assays were performed with GST-EPLIN-α (lane 1), GST-ΔN (lane 2), GST-αΔC (lane 3), GST-αΔC/LIM (lane 4), and GST-LIM (lane 5). (C) Schematic diagrams of EPLIN truncations used in the actin monomer pull-down assay are shown. (D) Actin filament pelleting assay. Samples containing 16 μM polymerized actin and 2 μM EPLIN-α were centrifuged at high speed. Control proteins (BSA, negative controls; α-actinin, positive control) were also used at 2 μM. The presence (+) or absence (−) of actin in the pelleting assay is denoted. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of control proteins and EPLIN-α, or its truncated derivatives, his-EPLINΔN/LIM and his-EPLINαΔC, are indicated by filled circles. (E) Binding of his-EPLIN-α to actin filament. His-EPLIN-α (0–8 μM) was mixed with 10 μM polymerized actin, followed by ultracentrifugation. Amounts of the free and bound his-EPLIN-α in the supernatant and pellet fractions were determined from a digitized Coomassie-stained gel.

Mentions: A recombinant full-length EPLIN-α GST-fusion protein purified from bacteria bound actin monomers in a pull-down assay (Fig. 3 A). Actin monomer binding was efficient and comparable to the control GST-VCA protein, pelleting ∼10% of the actin monomers. Truncated EPLIN containing either the NH2- or COOH-terminal region (GST-αΔC and GST-ΔN) also bound monomeric actin, but less efficiently than the full-length EPLIN-α (Fig. 3 B). GST-αΔC/LIM, lacking the central LIM domain, also bound to monomeric actin, but the LIM domain (GST-LIM) failed to bind monomeric actin. These findings indicate that EPLIN-α contains at least two actin-binding sites, one on each side of the centrally located LIM domain. Equivalent loading for the GST-fusion proteins in these experiments was confirmed by Coomassie staining.


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 binds directly to both G- and F-actin. (A) Actin monomer pull-down assay with GST-EPLIN. Glutathione-Sepharose beads carrying 2 μg of GST-EPLIN-α (lane 1), GST-VCA (lane 2), and GST (lane 3) were incubated with 50 nM G-actin in 0.5 ml. G-actin bound to the beads was assayed by immunoblotting with anti-actin antibodies (top). In lanes 4–6, varying amounts of G-actin (2.5–7.5% of the input) were included to estimate the amount of bound G-actin. A duplicate gel was stained with Coomassie blue to estimate the amounts of GST proteins present in each lane (bottom). GST-VCA contains a G-actin–binding verprolin (V) homology domain, a cofilin (C) homology domain, and an acidic segment (A) of bovine N-WASp. (B) Actin monomer pull-down assays were performed with GST-EPLIN-α (lane 1), GST-ΔN (lane 2), GST-αΔC (lane 3), GST-αΔC/LIM (lane 4), and GST-LIM (lane 5). (C) Schematic diagrams of EPLIN truncations used in the actin monomer pull-down assay are shown. (D) Actin filament pelleting assay. Samples containing 16 μM polymerized actin and 2 μM EPLIN-α were centrifuged at high speed. Control proteins (BSA, negative controls; α-actinin, positive control) were also used at 2 μM. The presence (+) or absence (−) of actin in the pelleting assay is denoted. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of control proteins and EPLIN-α, or its truncated derivatives, his-EPLINΔN/LIM and his-EPLINαΔC, are indicated by filled circles. (E) Binding of his-EPLIN-α to actin filament. His-EPLIN-α (0–8 μM) was mixed with 10 μM polymerized actin, followed by ultracentrifugation. Amounts of the free and bound his-EPLIN-α in the supernatant and pellet fractions were determined from a digitized Coomassie-stained gel.
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Related In: Results  -  Collection

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fig3: EPLIN binds directly to both G- and F-actin. (A) Actin monomer pull-down assay with GST-EPLIN. Glutathione-Sepharose beads carrying 2 μg of GST-EPLIN-α (lane 1), GST-VCA (lane 2), and GST (lane 3) were incubated with 50 nM G-actin in 0.5 ml. G-actin bound to the beads was assayed by immunoblotting with anti-actin antibodies (top). In lanes 4–6, varying amounts of G-actin (2.5–7.5% of the input) were included to estimate the amount of bound G-actin. A duplicate gel was stained with Coomassie blue to estimate the amounts of GST proteins present in each lane (bottom). GST-VCA contains a G-actin–binding verprolin (V) homology domain, a cofilin (C) homology domain, and an acidic segment (A) of bovine N-WASp. (B) Actin monomer pull-down assays were performed with GST-EPLIN-α (lane 1), GST-ΔN (lane 2), GST-αΔC (lane 3), GST-αΔC/LIM (lane 4), and GST-LIM (lane 5). (C) Schematic diagrams of EPLIN truncations used in the actin monomer pull-down assay are shown. (D) Actin filament pelleting assay. Samples containing 16 μM polymerized actin and 2 μM EPLIN-α were centrifuged at high speed. Control proteins (BSA, negative controls; α-actinin, positive control) were also used at 2 μM. The presence (+) or absence (−) of actin in the pelleting assay is denoted. The supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. Positions of control proteins and EPLIN-α, or its truncated derivatives, his-EPLINΔN/LIM and his-EPLINαΔC, are indicated by filled circles. (E) Binding of his-EPLIN-α to actin filament. His-EPLIN-α (0–8 μM) was mixed with 10 μM polymerized actin, followed by ultracentrifugation. Amounts of the free and bound his-EPLIN-α in the supernatant and pellet fractions were determined from a digitized Coomassie-stained gel.
Mentions: A recombinant full-length EPLIN-α GST-fusion protein purified from bacteria bound actin monomers in a pull-down assay (Fig. 3 A). Actin monomer binding was efficient and comparable to the control GST-VCA protein, pelleting ∼10% of the actin monomers. Truncated EPLIN containing either the NH2- or COOH-terminal region (GST-αΔC and GST-ΔN) also bound monomeric actin, but less efficiently than the full-length EPLIN-α (Fig. 3 B). GST-αΔC/LIM, lacking the central LIM domain, also bound to monomeric actin, but the LIM domain (GST-LIM) failed to bind monomeric actin. These findings indicate that EPLIN-α contains at least two actin-binding sites, one on each side of the centrally located LIM domain. Equivalent loading for the GST-fusion proteins in these experiments was confirmed by Coomassie staining.

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