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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 actin filament depolymerization and barbed-end elongation. (A) Time course of the depolymerization of actin filaments in the presence of EPLIN, capping protein, and α-actinin. Actin was polymerized with capping protein, EPLIN, or α-actinin, and was then diluted 20-fold into polymerization buffer to initiate depolymerization. The final protein concentration in the samples were 0.1 μM actin, ± 0.15 μM capping protein, ± 10 or 100 nM GST-EPLIN-α, and ± 0.25 μM α-actinin. The actin polymer concentration was monitored over time using pyrene fluorescence. (B) Time course of actin filament elongation from barbed ends provided by spectrin-actin seeds. Samples in polymerization buffer contained 2 μM actin (5% pyrene labeled), 9 nM spectrin-actin seeds, and ± 100 nM EPLIN-α. In the presence of capping protein, actin polymerization was inhibited by <90% (not depicted).
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fig5: Effect of EPLIN on actin filament depolymerization and barbed-end elongation. (A) Time course of the depolymerization of actin filaments in the presence of EPLIN, capping protein, and α-actinin. Actin was polymerized with capping protein, EPLIN, or α-actinin, and was then diluted 20-fold into polymerization buffer to initiate depolymerization. The final protein concentration in the samples were 0.1 μM actin, ± 0.15 μM capping protein, ± 10 or 100 nM GST-EPLIN-α, and ± 0.25 μM α-actinin. The actin polymer concentration was monitored over time using pyrene fluorescence. (B) Time course of actin filament elongation from barbed ends provided by spectrin-actin seeds. Samples in polymerization buffer contained 2 μM actin (5% pyrene labeled), 9 nM spectrin-actin seeds, and ± 100 nM EPLIN-α. In the presence of capping protein, actin polymerization was inhibited by <90% (not depicted).

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)

Effect of EPLIN on actin filament depolymerization and barbed-end elongation. (A) Time course of the depolymerization of actin filaments in the presence of EPLIN, capping protein, and α-actinin. Actin was polymerized with capping protein, EPLIN, or α-actinin, and was then diluted 20-fold into polymerization buffer to initiate depolymerization. The final protein concentration in the samples were 0.1 μM actin, ± 0.15 μM capping protein, ± 10 or 100 nM GST-EPLIN-α, and ± 0.25 μM α-actinin. The actin polymer concentration was monitored over time using pyrene fluorescence. (B) Time course of actin filament elongation from barbed ends provided by spectrin-actin seeds. Samples in polymerization buffer contained 2 μM actin (5% pyrene labeled), 9 nM spectrin-actin seeds, and ± 100 nM EPLIN-α. In the presence of capping protein, actin polymerization was inhibited by <90% (not depicted).
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Related In: Results  -  Collection

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fig5: Effect of EPLIN on actin filament depolymerization and barbed-end elongation. (A) Time course of the depolymerization of actin filaments in the presence of EPLIN, capping protein, and α-actinin. Actin was polymerized with capping protein, EPLIN, or α-actinin, and was then diluted 20-fold into polymerization buffer to initiate depolymerization. The final protein concentration in the samples were 0.1 μM actin, ± 0.15 μM capping protein, ± 10 or 100 nM GST-EPLIN-α, and ± 0.25 μM α-actinin. The actin polymer concentration was monitored over time using pyrene fluorescence. (B) Time course of actin filament elongation from barbed ends provided by spectrin-actin seeds. Samples in polymerization buffer contained 2 μM actin (5% pyrene labeled), 9 nM spectrin-actin seeds, and ± 100 nM EPLIN-α. In the presence of capping protein, actin polymerization was inhibited by <90% (not depicted).
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