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Local signaling by the EGF receptor.

Kempiak SJ, Yip SC, Backer JM, Segall JE - J. Cell Biol. (2003)

Bottom Line: We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression.The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function.Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

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Bead responses depend on actin polymerization and EGFR kinase. (A) MTLn3:EGFR cells were treated with 0.1% DMSO, 1 μM cytochalasin D, or 1 μM nocodazole for 1 min, or 1 μM PD153035 for 15 min. BSA beads or EGF beads were then added to the cells for 5 min followed by fixation and staining with rhodamine phalloidin. Total positive responses are reported. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. (B) EGF beads were added simultaneously with 5 nM EGF for three min. The cells were stained and fixed for F-actin. Arrow indicates location of bead in fluorescence image. Bar, 20 μm.
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fig2: Bead responses depend on actin polymerization and EGFR kinase. (A) MTLn3:EGFR cells were treated with 0.1% DMSO, 1 μM cytochalasin D, or 1 μM nocodazole for 1 min, or 1 μM PD153035 for 15 min. BSA beads or EGF beads were then added to the cells for 5 min followed by fixation and staining with rhodamine phalloidin. Total positive responses are reported. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. (B) EGF beads were added simultaneously with 5 nM EGF for three min. The cells were stained and fixed for F-actin. Arrow indicates location of bead in fluorescence image. Bar, 20 μm.

Mentions: To confirm that EGF receptor activity was responsible for the localized response rather than nonspecific clustering of other membrane proteins, MTLn3:EGFR cells were treated with PD153035, a drug that inhibits EGFR kinase activity, for 15 min before bead addition. This treatment significantly decreased the number of positive responses (Fig. 2 A), indicating that kinase activity of the EGFR was critical for generating localized responses to beads. Cytochalasin D completely inhibited the EGF bead response, whereas nocodazole treatment had no effect (Fig. 2 A). These data indicate that the increase in filamentous actin around EGF beads is not due to reorganization of preexisting actin but, rather, localized activation of actin polymerization and is independent of microtubule polymerization. The localized response was specific to binding of EGF to the EGFR, because the EGF bead response was completely inhibited when soluble EGF was added at a saturating concentration along with the EGF beads (Fig. 2 B). The soluble ligand stimulated the cell's EGF receptors, as indicated by the rim of filamentous actin at the edge of the cell, but blocked the access of the EGF beads to the receptors.


Local signaling by the EGF receptor.

Kempiak SJ, Yip SC, Backer JM, Segall JE - J. Cell Biol. (2003)

Bead responses depend on actin polymerization and EGFR kinase. (A) MTLn3:EGFR cells were treated with 0.1% DMSO, 1 μM cytochalasin D, or 1 μM nocodazole for 1 min, or 1 μM PD153035 for 15 min. BSA beads or EGF beads were then added to the cells for 5 min followed by fixation and staining with rhodamine phalloidin. Total positive responses are reported. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. (B) EGF beads were added simultaneously with 5 nM EGF for three min. The cells were stained and fixed for F-actin. Arrow indicates location of bead in fluorescence image. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172819&req=5

fig2: Bead responses depend on actin polymerization and EGFR kinase. (A) MTLn3:EGFR cells were treated with 0.1% DMSO, 1 μM cytochalasin D, or 1 μM nocodazole for 1 min, or 1 μM PD153035 for 15 min. BSA beads or EGF beads were then added to the cells for 5 min followed by fixation and staining with rhodamine phalloidin. Total positive responses are reported. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. (B) EGF beads were added simultaneously with 5 nM EGF for three min. The cells were stained and fixed for F-actin. Arrow indicates location of bead in fluorescence image. Bar, 20 μm.
Mentions: To confirm that EGF receptor activity was responsible for the localized response rather than nonspecific clustering of other membrane proteins, MTLn3:EGFR cells were treated with PD153035, a drug that inhibits EGFR kinase activity, for 15 min before bead addition. This treatment significantly decreased the number of positive responses (Fig. 2 A), indicating that kinase activity of the EGFR was critical for generating localized responses to beads. Cytochalasin D completely inhibited the EGF bead response, whereas nocodazole treatment had no effect (Fig. 2 A). These data indicate that the increase in filamentous actin around EGF beads is not due to reorganization of preexisting actin but, rather, localized activation of actin polymerization and is independent of microtubule polymerization. The localized response was specific to binding of EGF to the EGFR, because the EGF bead response was completely inhibited when soluble EGF was added at a saturating concentration along with the EGF beads (Fig. 2 B). The soluble ligand stimulated the cell's EGF receptors, as indicated by the rim of filamentous actin at the edge of the cell, but blocked the access of the EGF beads to the receptors.

Bottom Line: We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression.The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function.Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.

Show MeSH
Related in: MedlinePlus