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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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The Arp2/3 complex and cofilin work synergistically to create the EGF bead response. (A) MTLn3:EGFR cells transfected with control (white), p34 (dark gray), cofilin (stripes), or both p34 and cofilin siRNA (light gray) for 4 h. Cells were then cultured for 24–48 h before being analyzed. Cells were stimulated with EGF-coated beads for 80 s, fixed, and stained for rhodamine phalloidin, and the cell response was analyzed. Data represent the mean ± SEM from >180 cells in three separate experiments. (B) Lysates from cells treated with siRNA as in A were blotted for cofilin and p34 protein expression levels.
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fig5: The Arp2/3 complex and cofilin work synergistically to create the EGF bead response. (A) MTLn3:EGFR cells transfected with control (white), p34 (dark gray), cofilin (stripes), or both p34 and cofilin siRNA (light gray) for 4 h. Cells were then cultured for 24–48 h before being analyzed. Cells were stimulated with EGF-coated beads for 80 s, fixed, and stained for rhodamine phalloidin, and the cell response was analyzed. Data represent the mean ± SEM from >180 cells in three separate experiments. (B) Lysates from cells treated with siRNA as in A were blotted for cofilin and p34 protein expression levels.

Mentions: To identify the mechanism of localized actin polymerization, we evaluated the roles of cofilin and the Arp2/3 complex. Actin polymerization at the leading edge of MTLn3 cells in response to soluble EGF requires both Arp2/3 complex (Bailly et al., 2001) and cofilin (Chan et al., 2000) activity in vitro as well as in vivo (DesMarais, V., F. Macaluso, J. Condeelis, and M. Bailly, personal communication). We used siRNA to reduce the expression levels of p34 and cofilin individually or together (Fig. 5). When transfected into cells, the siRNAs nearly abolished the expression of the appropriate proteins whereas control siRNA had no effect. Knock-down of either p34 or cofilin individually decreased the protrusion response and the total positive response by half. When both constructs were expressed in the same cell line, the protrusion response was practically ablated, and total positive response dropped by ∼80%. Therefore, both Arp2/3 complex and cofilin can mediate localized actin polymerization responses.


Local signaling by the EGF receptor.

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

The Arp2/3 complex and cofilin work synergistically to create the EGF bead response. (A) MTLn3:EGFR cells transfected with control (white), p34 (dark gray), cofilin (stripes), or both p34 and cofilin siRNA (light gray) for 4 h. Cells were then cultured for 24–48 h before being analyzed. Cells were stimulated with EGF-coated beads for 80 s, fixed, and stained for rhodamine phalloidin, and the cell response was analyzed. Data represent the mean ± SEM from >180 cells in three separate experiments. (B) Lysates from cells treated with siRNA as in A were blotted for cofilin and p34 protein expression levels.
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Related In: Results  -  Collection

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

fig5: The Arp2/3 complex and cofilin work synergistically to create the EGF bead response. (A) MTLn3:EGFR cells transfected with control (white), p34 (dark gray), cofilin (stripes), or both p34 and cofilin siRNA (light gray) for 4 h. Cells were then cultured for 24–48 h before being analyzed. Cells were stimulated with EGF-coated beads for 80 s, fixed, and stained for rhodamine phalloidin, and the cell response was analyzed. Data represent the mean ± SEM from >180 cells in three separate experiments. (B) Lysates from cells treated with siRNA as in A were blotted for cofilin and p34 protein expression levels.
Mentions: To identify the mechanism of localized actin polymerization, we evaluated the roles of cofilin and the Arp2/3 complex. Actin polymerization at the leading edge of MTLn3 cells in response to soluble EGF requires both Arp2/3 complex (Bailly et al., 2001) and cofilin (Chan et al., 2000) activity in vitro as well as in vivo (DesMarais, V., F. Macaluso, J. Condeelis, and M. Bailly, personal communication). We used siRNA to reduce the expression levels of p34 and cofilin individually or together (Fig. 5). When transfected into cells, the siRNAs nearly abolished the expression of the appropriate proteins whereas control siRNA had no effect. Knock-down of either p34 or cofilin individually decreased the protrusion response and the total positive response by half. When both constructs were expressed in the same cell line, the protrusion response was practically ablated, and total positive response dropped by ∼80%. Therefore, both Arp2/3 complex and cofilin can mediate localized actin polymerization responses.

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