Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of EPLIN increases actin filaments in MCF-7 cells. (A, B, D, and E) Expression of Flag-EPLIN-α or -β was induced by removing doxycycline from culture media. 72 h after protein induction, cells were fixed and stained with rhodamine-phalloidin to visualize actin filaments. A and D, − induction; B and E, + induction. Digital images were captured at the same exposure time (248 ms for A and B; 308 ms for D and E). (C and F) The levels of EPLIN overexpression in MCF-7 cells were estimated. Varying amounts (2.5–12.5 μg) of protein lysates from MCF-7 cells before and after protein induction, human mammary epithelial cells (HMEC; EPLIN-α positive), and BeWo choriocarcinoma cells (EPLIN-β positive) were fractionated by SDS-PAGE and then immunoblotted with anti-EPLIN antisera. To estimate the absolute level of expression, known amounts (0.5–10 ng) of purified his-EPLIN-α protein were also included in the immunoblot. (G and H) Flag-EPLIN-α or -β expressing MCF-7 cells were stained with rhodamine-phalloidin and the M2 anti-Flag mAb to stain the induced proteins. The increase in actin filaments correlated with the amount of EPLIN expressed in these cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172667&req=5

fig1: Expression of EPLIN increases actin filaments in MCF-7 cells. (A, B, D, and E) Expression of Flag-EPLIN-α or -β was induced by removing doxycycline from culture media. 72 h after protein induction, cells were fixed and stained with rhodamine-phalloidin to visualize actin filaments. A and D, − induction; B and E, + induction. Digital images were captured at the same exposure time (248 ms for A and B; 308 ms for D and E). (C and F) The levels of EPLIN overexpression in MCF-7 cells were estimated. Varying amounts (2.5–12.5 μg) of protein lysates from MCF-7 cells before and after protein induction, human mammary epithelial cells (HMEC; EPLIN-α positive), and BeWo choriocarcinoma cells (EPLIN-β positive) were fractionated by SDS-PAGE and then immunoblotted with anti-EPLIN antisera. To estimate the absolute level of expression, known amounts (0.5–10 ng) of purified his-EPLIN-α protein were also included in the immunoblot. (G and H) Flag-EPLIN-α or -β expressing MCF-7 cells were stained with rhodamine-phalloidin and the M2 anti-Flag mAb to stain the induced proteins. The increase in actin filaments correlated with the amount of EPLIN expressed in these cells.

Mentions: To test if EPLIN modifies actin organization in the cell, we expressed EPLIN in MCF-7 breast carcinoma cells using a tetracycline-regulated system. MCF-7 cells do not express EPLIN-α, but express EPLIN-β at a low level (Fig. 1 C, lane 9). Induction of EPLIN expression increased the number of actin stress fibers and the intensity with which they stained with rhodamine-phalloidin (Fig. 1 A, B, D, and E). Expression of EPLIN-α in MCF-7 cells increased the fluorescence intensity of rhodamine-phalloidin staining 2.1-fold, whereas expression of EPLIN-β increased actin staining 3.7-fold (see Materials and methods). The levels of EPLIN-α and -β overexpression in MCF-7 cells were equivalent to the levels of endogenous protein in HMEC and BeWo choriocarcinoma cells, respectively (Fig. 1, C and F). Staining for the induced EPLIN using anti-Flag antibodies revealed a positive correlation between the levels of EPLIN expression and the amount of stress fibers (Fig. 1, G and H).


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)

Expression of EPLIN increases actin filaments in MCF-7 cells. (A, B, D, and E) Expression of Flag-EPLIN-α or -β was induced by removing doxycycline from culture media. 72 h after protein induction, cells were fixed and stained with rhodamine-phalloidin to visualize actin filaments. A and D, − induction; B and E, + induction. Digital images were captured at the same exposure time (248 ms for A and B; 308 ms for D and E). (C and F) The levels of EPLIN overexpression in MCF-7 cells were estimated. Varying amounts (2.5–12.5 μg) of protein lysates from MCF-7 cells before and after protein induction, human mammary epithelial cells (HMEC; EPLIN-α positive), and BeWo choriocarcinoma cells (EPLIN-β positive) were fractionated by SDS-PAGE and then immunoblotted with anti-EPLIN antisera. To estimate the absolute level of expression, known amounts (0.5–10 ng) of purified his-EPLIN-α protein were also included in the immunoblot. (G and H) Flag-EPLIN-α or -β expressing MCF-7 cells were stained with rhodamine-phalloidin and the M2 anti-Flag mAb to stain the induced proteins. The increase in actin filaments correlated with the amount of EPLIN expressed in these cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Expression of EPLIN increases actin filaments in MCF-7 cells. (A, B, D, and E) Expression of Flag-EPLIN-α or -β was induced by removing doxycycline from culture media. 72 h after protein induction, cells were fixed and stained with rhodamine-phalloidin to visualize actin filaments. A and D, − induction; B and E, + induction. Digital images were captured at the same exposure time (248 ms for A and B; 308 ms for D and E). (C and F) The levels of EPLIN overexpression in MCF-7 cells were estimated. Varying amounts (2.5–12.5 μg) of protein lysates from MCF-7 cells before and after protein induction, human mammary epithelial cells (HMEC; EPLIN-α positive), and BeWo choriocarcinoma cells (EPLIN-β positive) were fractionated by SDS-PAGE and then immunoblotted with anti-EPLIN antisera. To estimate the absolute level of expression, known amounts (0.5–10 ng) of purified his-EPLIN-α protein were also included in the immunoblot. (G and H) Flag-EPLIN-α or -β expressing MCF-7 cells were stained with rhodamine-phalloidin and the M2 anti-Flag mAb to stain the induced proteins. The increase in actin filaments correlated with the amount of EPLIN expressed in these cells.
Mentions: To test if EPLIN modifies actin organization in the cell, we expressed EPLIN in MCF-7 breast carcinoma cells using a tetracycline-regulated system. MCF-7 cells do not express EPLIN-α, but express EPLIN-β at a low level (Fig. 1 C, lane 9). Induction of EPLIN expression increased the number of actin stress fibers and the intensity with which they stained with rhodamine-phalloidin (Fig. 1 A, B, D, and E). Expression of EPLIN-α in MCF-7 cells increased the fluorescence intensity of rhodamine-phalloidin staining 2.1-fold, whereas expression of EPLIN-β increased actin staining 3.7-fold (see Materials and methods). The levels of EPLIN-α and -β overexpression in MCF-7 cells were equivalent to the levels of endogenous protein in HMEC and BeWo choriocarcinoma cells, respectively (Fig. 1, C and F). Staining for the induced EPLIN using anti-Flag antibodies revealed a positive correlation between the levels of EPLIN expression and the amount of stress fibers (Fig. 1, G and H).

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