Limits...
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.

Show MeSH

Related in: MedlinePlus

Localization of signaling proteins around EGF beads. (A) The relative increase in signal for the indicated molecule near the bead (5 min after application), relative to plasma membranes far from the bead in the same plane of focus, is given. Data are mean ± SEM from >33 cells in two or more separate experiments. (B) The areas of increased intensity of the samples measured in A were determined, and a radius calculated, assuming a circular shape. Means and SEM from >33 cells in two or more experiments are shown. (C) Comparison of phase, phosphotyrosine (pTyr, red), F-actin (blue), and phosphoAkt (pAkt, green) distributions in one cell stimulated with an EGF bead. Bar, 2 μm. (D) Global ERK activation. Cells were stimulated with buffer (left), 10 nM soluble EGF (middle), or EGF beads (right) for 5 min and then fixed and stained for phosphoERK (fluorescence on top, phase images on bottom). Beads are phase bright in the right hand phase image. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172819&req=5

fig3: Localization of signaling proteins around EGF beads. (A) The relative increase in signal for the indicated molecule near the bead (5 min after application), relative to plasma membranes far from the bead in the same plane of focus, is given. Data are mean ± SEM from >33 cells in two or more separate experiments. (B) The areas of increased intensity of the samples measured in A were determined, and a radius calculated, assuming a circular shape. Means and SEM from >33 cells in two or more experiments are shown. (C) Comparison of phase, phosphotyrosine (pTyr, red), F-actin (blue), and phosphoAkt (pAkt, green) distributions in one cell stimulated with an EGF bead. Bar, 2 μm. (D) Global ERK activation. Cells were stimulated with buffer (left), 10 nM soluble EGF (middle), or EGF beads (right) for 5 min and then fixed and stained for phosphoERK (fluorescence on top, phase images on bottom). Beads are phase bright in the right hand phase image. Bar, 10 μm.

Mentions: Given the published data that EGFR activation can spread throughout the cell (Verveer et al., 2000; Sawano et al., 2002), we wished to identify which signaling pathways remained localized with the filamentous actin response (Fig. 3 A). To determine if the localized response depended on an increased local concentration of EGFR in response to the bead, MTLn3 cells overexpressing an EGFR–GFP fusion protein (Bailly et al., 2000) were used to observe the receptor levels at the bead site. When exposed to EGF beads, GFP fluorescence was not increased at the site of the bead (Fig. 3 A, EGFR:GFP column). Thus, EGF beads could induce a localized response to EGF without increasing the local density of EGFR, although EGF may be released from the bead upon binding to receptor. Both phosphotyrosine staining and phosphoErbB2 were increased near the bead site. When MTLn3 cells are stimulated with EGF, the Arp2/3 complex has been shown to accumulate at the leading edge of a cell (Bailly et al., 2001). N-WASP, an activator of the Arp2/3 complex, and p34, a subunit of the Arp2/3 complex, were both increased near the bead compared with levels at the edge of the cell. Cofilin, an actin-severing protein, which is near the leading edge of lamellipodia (Chan et al., 2000) also localized to the bead site (Fig. 3 A). These data are consistent with data indicating localized accumulation of shc around EGF beads in A431 cells (Brock and Jovin, 2001). However, FAK and vinculin, proteins associated with focal adhesions (Geiger et al., 2001), did not localize to the source of the bead, arguing against an adhesion or focal contact mechanism for generating the local actin polymerization.


Local signaling by the EGF receptor.

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

Localization of signaling proteins around EGF beads. (A) The relative increase in signal for the indicated molecule near the bead (5 min after application), relative to plasma membranes far from the bead in the same plane of focus, is given. Data are mean ± SEM from >33 cells in two or more separate experiments. (B) The areas of increased intensity of the samples measured in A were determined, and a radius calculated, assuming a circular shape. Means and SEM from >33 cells in two or more experiments are shown. (C) Comparison of phase, phosphotyrosine (pTyr, red), F-actin (blue), and phosphoAkt (pAkt, green) distributions in one cell stimulated with an EGF bead. Bar, 2 μm. (D) Global ERK activation. Cells were stimulated with buffer (left), 10 nM soluble EGF (middle), or EGF beads (right) for 5 min and then fixed and stained for phosphoERK (fluorescence on top, phase images on bottom). Beads are phase bright in the right hand phase image. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Localization of signaling proteins around EGF beads. (A) The relative increase in signal for the indicated molecule near the bead (5 min after application), relative to plasma membranes far from the bead in the same plane of focus, is given. Data are mean ± SEM from >33 cells in two or more separate experiments. (B) The areas of increased intensity of the samples measured in A were determined, and a radius calculated, assuming a circular shape. Means and SEM from >33 cells in two or more experiments are shown. (C) Comparison of phase, phosphotyrosine (pTyr, red), F-actin (blue), and phosphoAkt (pAkt, green) distributions in one cell stimulated with an EGF bead. Bar, 2 μm. (D) Global ERK activation. Cells were stimulated with buffer (left), 10 nM soluble EGF (middle), or EGF beads (right) for 5 min and then fixed and stained for phosphoERK (fluorescence on top, phase images on bottom). Beads are phase bright in the right hand phase image. Bar, 10 μm.
Mentions: Given the published data that EGFR activation can spread throughout the cell (Verveer et al., 2000; Sawano et al., 2002), we wished to identify which signaling pathways remained localized with the filamentous actin response (Fig. 3 A). To determine if the localized response depended on an increased local concentration of EGFR in response to the bead, MTLn3 cells overexpressing an EGFR–GFP fusion protein (Bailly et al., 2000) were used to observe the receptor levels at the bead site. When exposed to EGF beads, GFP fluorescence was not increased at the site of the bead (Fig. 3 A, EGFR:GFP column). Thus, EGF beads could induce a localized response to EGF without increasing the local density of EGFR, although EGF may be released from the bead upon binding to receptor. Both phosphotyrosine staining and phosphoErbB2 were increased near the bead site. When MTLn3 cells are stimulated with EGF, the Arp2/3 complex has been shown to accumulate at the leading edge of a cell (Bailly et al., 2001). N-WASP, an activator of the Arp2/3 complex, and p34, a subunit of the Arp2/3 complex, were both increased near the bead compared with levels at the edge of the cell. Cofilin, an actin-severing protein, which is near the leading edge of lamellipodia (Chan et al., 2000) also localized to the bead site (Fig. 3 A). These data are consistent with data indicating localized accumulation of shc around EGF beads in A431 cells (Brock and Jovin, 2001). However, FAK and vinculin, proteins associated with focal adhesions (Geiger et al., 2001), did not localize to the source of the bead, arguing against an adhesion or focal contact mechanism for generating the local actin polymerization.

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