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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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Responses to EGF-coated beads. (A) MTLn3:EGFR cells were imaged before (first frame, 0.00) and after the addition of EGF beads (1.00 to 17 min 30 s). Arrows indicate beads that induced localized phase densities in subsequent images and the arrowhead indicates a bead that induced a protrusion (Video 1). Bar, 20 μm. (B) Projections from a confocal z series of cells stimulated for 5 min by EGF beads. F-actin is in red and the bead in green. (Top) Confocal slice through the bead region. (Bottom) Side view projection. Bar, 10 μm. (C) Projections from a confocal z series of a protrusion induced by an EGF bead. Bar, 10 μm. See Videos 2 and 3 for rotations of B and C. (D) Kinetics of actin polymerization. The dashed curve represents all positive responses as indicated by increased rhodamine phalloidin staining (including protrusions). The solid curve represents protrusions only. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. All videos are available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1.
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fig1: Responses to EGF-coated beads. (A) MTLn3:EGFR cells were imaged before (first frame, 0.00) and after the addition of EGF beads (1.00 to 17 min 30 s). Arrows indicate beads that induced localized phase densities in subsequent images and the arrowhead indicates a bead that induced a protrusion (Video 1). Bar, 20 μm. (B) Projections from a confocal z series of cells stimulated for 5 min by EGF beads. F-actin is in red and the bead in green. (Top) Confocal slice through the bead region. (Bottom) Side view projection. Bar, 10 μm. (C) Projections from a confocal z series of a protrusion induced by an EGF bead. Bar, 10 μm. See Videos 2 and 3 for rotations of B and C. (D) Kinetics of actin polymerization. The dashed curve represents all positive responses as indicated by increased rhodamine phalloidin staining (including protrusions). The solid curve represents protrusions only. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. All videos are available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1.

Mentions: We initially used metastatic rat mammary adenocarcinoma cells (MTLn3-EGFR) overexpressing the EGF receptor (>105 EGFR per cell) to identify localized cellular responses. MTLn3-EGFR cells are chemotactic to spatial gradients of EGF, generating actin-rich lamellipods that extend toward higher concentrations of EGF (Bailly et al., 2000). We have used streptavidin-coupled magnetic beads bound to biotin-labeled EGF in order to minimize release of EGF from the bead. A magnet was used to pull the beads rapidly onto the surfaces of the cells. By performing time-lapse analysis, we observed two types of cellular responses to the beads (Fig. 1 A; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1). The predominant response was the production of a phase-dense ring or ruffling around the bead (beads marked by arrows in Fig. 1 A). However, >30% of the responses were protrusions with the bead at the tip: in the center cell of Fig. 1 A (bead marked by arrowhead), a protrusion in the form of a helix developed over a ten-min period after contacting the cell (see Video 1).


Local signaling by the EGF receptor.

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

Responses to EGF-coated beads. (A) MTLn3:EGFR cells were imaged before (first frame, 0.00) and after the addition of EGF beads (1.00 to 17 min 30 s). Arrows indicate beads that induced localized phase densities in subsequent images and the arrowhead indicates a bead that induced a protrusion (Video 1). Bar, 20 μm. (B) Projections from a confocal z series of cells stimulated for 5 min by EGF beads. F-actin is in red and the bead in green. (Top) Confocal slice through the bead region. (Bottom) Side view projection. Bar, 10 μm. (C) Projections from a confocal z series of a protrusion induced by an EGF bead. Bar, 10 μm. See Videos 2 and 3 for rotations of B and C. (D) Kinetics of actin polymerization. The dashed curve represents all positive responses as indicated by increased rhodamine phalloidin staining (including protrusions). The solid curve represents protrusions only. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. All videos are available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Responses to EGF-coated beads. (A) MTLn3:EGFR cells were imaged before (first frame, 0.00) and after the addition of EGF beads (1.00 to 17 min 30 s). Arrows indicate beads that induced localized phase densities in subsequent images and the arrowhead indicates a bead that induced a protrusion (Video 1). Bar, 20 μm. (B) Projections from a confocal z series of cells stimulated for 5 min by EGF beads. F-actin is in red and the bead in green. (Top) Confocal slice through the bead region. (Bottom) Side view projection. Bar, 10 μm. (C) Projections from a confocal z series of a protrusion induced by an EGF bead. Bar, 10 μm. See Videos 2 and 3 for rotations of B and C. (D) Kinetics of actin polymerization. The dashed curve represents all positive responses as indicated by increased rhodamine phalloidin staining (including protrusions). The solid curve represents protrusions only. Data represent the mean ± SEM of three experiments; n = 50 for each experiment. All videos are available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1.
Mentions: We initially used metastatic rat mammary adenocarcinoma cells (MTLn3-EGFR) overexpressing the EGF receptor (>105 EGFR per cell) to identify localized cellular responses. MTLn3-EGFR cells are chemotactic to spatial gradients of EGF, generating actin-rich lamellipods that extend toward higher concentrations of EGF (Bailly et al., 2000). We have used streptavidin-coupled magnetic beads bound to biotin-labeled EGF in order to minimize release of EGF from the bead. A magnet was used to pull the beads rapidly onto the surfaces of the cells. By performing time-lapse analysis, we observed two types of cellular responses to the beads (Fig. 1 A; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200303144/DC1). The predominant response was the production of a phase-dense ring or ruffling around the bead (beads marked by arrows in Fig. 1 A). However, >30% of the responses were protrusions with the bead at the tip: in the center cell of Fig. 1 A (bead marked by arrowhead), a protrusion in the form of a helix developed over a ten-min period after contacting the cell (see Video 1).

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