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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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Effect of EPLIN on the morphology of filaments formed by Arp2/3 complex. (A and B) Fluorescence micrographs of actin filaments nucleated by Arp2/3 complex in the absence (A) or presence (B) of EPLIN. Samples contained 4 μM G-actin (5% pyrene labeled), 30 nM Arp2/3 complex, 100 nM Scar-WA domain, and varying concentrations of GST-EPLIN-α (0–900 nM) in polymerization buffer. Polymerization was performed in 4 μM rhodamine-phalloidin, which stabilizes branched structure by inhibiting phosphate release from ADP-Pi-actin filaments and allows visualization of filaments by fluorescence microscopy. Polymerization was allowed to proceed for 2 min before being quenched by dilution. (C) Quantitation of branched and unbranched filaments. The chi-squared test was used to calculate the p-values.
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fig7: Effect of EPLIN on the morphology of filaments formed by Arp2/3 complex. (A and B) Fluorescence micrographs of actin filaments nucleated by Arp2/3 complex in the absence (A) or presence (B) of EPLIN. Samples contained 4 μM G-actin (5% pyrene labeled), 30 nM Arp2/3 complex, 100 nM Scar-WA domain, and varying concentrations of GST-EPLIN-α (0–900 nM) in polymerization buffer. Polymerization was performed in 4 μM rhodamine-phalloidin, which stabilizes branched structure by inhibiting phosphate release from ADP-Pi-actin filaments and allows visualization of filaments by fluorescence microscopy. Polymerization was allowed to proceed for 2 min before being quenched by dilution. (C) Quantitation of branched and unbranched filaments. The chi-squared test was used to calculate the p-values.

Mentions: If EPLIN delays actin filament nucleation mediated by Arp2/3 complex by binding and sequestering actin filaments, the formation of branched filaments should be inhibited. By fluorescence microscopy, we measured branching of the products produced by Arp2/3 complex. After 2 min of polymerization in the presence of activated Arp2/3 complex, most (88%) of the products were short, branched filaments (Fig. 7). EPLIN-α reduced the fraction of branched filaments in a concentration-dependent fashion. Similar to the pyrene fluorescence assay, the inhibition occurred at nanomolar concentrations of EPLIN-α, reducing the fraction of branched filaments to 62% at 9 nM, 55% at 90 nM, and 17% at 900 nM. As seen in the kinetic studies, the inhibition of branch formation was transient; even with 900 nM EPLIN-α, the frequency of branches increased to 42% after 15 min of polymerization.


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)

Effect of EPLIN on the morphology of filaments formed by Arp2/3 complex. (A and B) Fluorescence micrographs of actin filaments nucleated by Arp2/3 complex in the absence (A) or presence (B) of EPLIN. Samples contained 4 μM G-actin (5% pyrene labeled), 30 nM Arp2/3 complex, 100 nM Scar-WA domain, and varying concentrations of GST-EPLIN-α (0–900 nM) in polymerization buffer. Polymerization was performed in 4 μM rhodamine-phalloidin, which stabilizes branched structure by inhibiting phosphate release from ADP-Pi-actin filaments and allows visualization of filaments by fluorescence microscopy. Polymerization was allowed to proceed for 2 min before being quenched by dilution. (C) Quantitation of branched and unbranched filaments. The chi-squared test was used to calculate the p-values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172667&req=5

fig7: Effect of EPLIN on the morphology of filaments formed by Arp2/3 complex. (A and B) Fluorescence micrographs of actin filaments nucleated by Arp2/3 complex in the absence (A) or presence (B) of EPLIN. Samples contained 4 μM G-actin (5% pyrene labeled), 30 nM Arp2/3 complex, 100 nM Scar-WA domain, and varying concentrations of GST-EPLIN-α (0–900 nM) in polymerization buffer. Polymerization was performed in 4 μM rhodamine-phalloidin, which stabilizes branched structure by inhibiting phosphate release from ADP-Pi-actin filaments and allows visualization of filaments by fluorescence microscopy. Polymerization was allowed to proceed for 2 min before being quenched by dilution. (C) Quantitation of branched and unbranched filaments. The chi-squared test was used to calculate the p-values.
Mentions: If EPLIN delays actin filament nucleation mediated by Arp2/3 complex by binding and sequestering actin filaments, the formation of branched filaments should be inhibited. By fluorescence microscopy, we measured branching of the products produced by Arp2/3 complex. After 2 min of polymerization in the presence of activated Arp2/3 complex, most (88%) of the products were short, branched filaments (Fig. 7). EPLIN-α reduced the fraction of branched filaments in a concentration-dependent fashion. Similar to the pyrene fluorescence assay, the inhibition occurred at nanomolar concentrations of EPLIN-α, reducing the fraction of branched filaments to 62% at 9 nM, 55% at 90 nM, and 17% at 900 nM. As seen in the kinetic studies, the inhibition of branch formation was transient; even with 900 nM EPLIN-α, the frequency of branches increased to 42% after 15 min of polymerization.

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