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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 inhibits actin nucleation by Arp2/3 complex. (A) The time course of actin polymerization was monitored by pyrene fluorescence. Samples contained 4 μM G-actin (5% pyrene labeled), ± 15 nM Arp2/3 complex, or ± 300 nM Scar-WA domain, ± varying concentrations of GST-EPLIN-α in polymerization buffer. (B) Dependence of the lag time and concentration of ends produced by Arp2/3 complex on the concentration of EPLIN-α. The polymerization lag time was defined as the time required to achieve 10% of the maximum fluorescence. The concentration of barbed ends was calculated from elongation rate (measured by the rate of polymerization where 80% of monomers were polymerized) using the equation: [barbed ends] = elongation rate/(k+ [actin monomer]), where k+ = 10 μM−1 s−1. At GST-EPLIN-α concentrations >105 nM, polymerization was incomplete in the time course of the experiment, disallowing determination of the lag time or the concentration of barbed ends. (C) The effect of EPLIN on the ability of preformed actin filaments to stimulate nucleation by Arp2/3 complex. Samples in polymerization buffer contained 2 μM G-actin (5% pyrene labeled), 15 nM Arp2/3 complex, 300 nM Scar-WA domain, and 8 μM profilin-I, ± 100 nM actin filament, ± 10 or 50 nM GST-EPLIN-α. Profilin-I was added to accentuate the polymerization lag time.
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fig6: EPLIN inhibits actin nucleation by Arp2/3 complex. (A) The time course of actin polymerization was monitored by pyrene fluorescence. Samples contained 4 μM G-actin (5% pyrene labeled), ± 15 nM Arp2/3 complex, or ± 300 nM Scar-WA domain, ± varying concentrations of GST-EPLIN-α in polymerization buffer. (B) Dependence of the lag time and concentration of ends produced by Arp2/3 complex on the concentration of EPLIN-α. The polymerization lag time was defined as the time required to achieve 10% of the maximum fluorescence. The concentration of barbed ends was calculated from elongation rate (measured by the rate of polymerization where 80% of monomers were polymerized) using the equation: [barbed ends] = elongation rate/(k+ [actin monomer]), where k+ = 10 μM−1 s−1. At GST-EPLIN-α concentrations >105 nM, polymerization was incomplete in the time course of the experiment, disallowing determination of the lag time or the concentration of barbed ends. (C) The effect of EPLIN on the ability of preformed actin filaments to stimulate nucleation by Arp2/3 complex. Samples in polymerization buffer contained 2 μM G-actin (5% pyrene labeled), 15 nM Arp2/3 complex, 300 nM Scar-WA domain, and 8 μM profilin-I, ± 100 nM actin filament, ± 10 or 50 nM GST-EPLIN-α. Profilin-I was added to accentuate the polymerization lag time.

Mentions: We used the fluorescence of pyrene-labeled actin to monitor polymerization and to test if EPLIN affects actin assembly or disassembly. The time course of spontaneous polymerization was the same in the presence or absence of EPLIN-α (see Fig. 6). On the other hand, EPLIN significantly delayed actin filament depolymerization (Fig. 5 A). When diluted below the critical concentration, actin filaments depolymerized with a half-time of 7 min. EPLIN-α slowed depolymerization in a concentration-dependent fashion. Addition and loss of actin monomers occurs exclusively at the filament ends, with the barbed ends displaying higher rates of turnover (Pollard et al., 2000). We tested the possibility that EPLIN-α stabilizes filaments by capping barbed ends (Fig. 5 B). The rate of barbed end elongation from spectrin-actin seeds was not affected by EPLIN-α, indicating that the EPLIN-α does not cap barbed ends.


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 inhibits actin nucleation by Arp2/3 complex. (A) The time course of actin polymerization was monitored by pyrene fluorescence. Samples contained 4 μM G-actin (5% pyrene labeled), ± 15 nM Arp2/3 complex, or ± 300 nM Scar-WA domain, ± varying concentrations of GST-EPLIN-α in polymerization buffer. (B) Dependence of the lag time and concentration of ends produced by Arp2/3 complex on the concentration of EPLIN-α. The polymerization lag time was defined as the time required to achieve 10% of the maximum fluorescence. The concentration of barbed ends was calculated from elongation rate (measured by the rate of polymerization where 80% of monomers were polymerized) using the equation: [barbed ends] = elongation rate/(k+ [actin monomer]), where k+ = 10 μM−1 s−1. At GST-EPLIN-α concentrations >105 nM, polymerization was incomplete in the time course of the experiment, disallowing determination of the lag time or the concentration of barbed ends. (C) The effect of EPLIN on the ability of preformed actin filaments to stimulate nucleation by Arp2/3 complex. Samples in polymerization buffer contained 2 μM G-actin (5% pyrene labeled), 15 nM Arp2/3 complex, 300 nM Scar-WA domain, and 8 μM profilin-I, ± 100 nM actin filament, ± 10 or 50 nM GST-EPLIN-α. Profilin-I was added to accentuate the polymerization lag time.
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Related In: Results  -  Collection

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fig6: EPLIN inhibits actin nucleation by Arp2/3 complex. (A) The time course of actin polymerization was monitored by pyrene fluorescence. Samples contained 4 μM G-actin (5% pyrene labeled), ± 15 nM Arp2/3 complex, or ± 300 nM Scar-WA domain, ± varying concentrations of GST-EPLIN-α in polymerization buffer. (B) Dependence of the lag time and concentration of ends produced by Arp2/3 complex on the concentration of EPLIN-α. The polymerization lag time was defined as the time required to achieve 10% of the maximum fluorescence. The concentration of barbed ends was calculated from elongation rate (measured by the rate of polymerization where 80% of monomers were polymerized) using the equation: [barbed ends] = elongation rate/(k+ [actin monomer]), where k+ = 10 μM−1 s−1. At GST-EPLIN-α concentrations >105 nM, polymerization was incomplete in the time course of the experiment, disallowing determination of the lag time or the concentration of barbed ends. (C) The effect of EPLIN on the ability of preformed actin filaments to stimulate nucleation by Arp2/3 complex. Samples in polymerization buffer contained 2 μM G-actin (5% pyrene labeled), 15 nM Arp2/3 complex, 300 nM Scar-WA domain, and 8 μM profilin-I, ± 100 nM actin filament, ± 10 or 50 nM GST-EPLIN-α. Profilin-I was added to accentuate the polymerization lag time.
Mentions: We used the fluorescence of pyrene-labeled actin to monitor polymerization and to test if EPLIN affects actin assembly or disassembly. The time course of spontaneous polymerization was the same in the presence or absence of EPLIN-α (see Fig. 6). On the other hand, EPLIN significantly delayed actin filament depolymerization (Fig. 5 A). When diluted below the critical concentration, actin filaments depolymerized with a half-time of 7 min. EPLIN-α slowed depolymerization in a concentration-dependent fashion. Addition and loss of actin monomers occurs exclusively at the filament ends, with the barbed ends displaying higher rates of turnover (Pollard et al., 2000). We tested the possibility that EPLIN-α stabilizes filaments by capping barbed ends (Fig. 5 B). The rate of barbed end elongation from spectrin-actin seeds was not affected by EPLIN-α, indicating that the EPLIN-α does not cap barbed ends.

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