Epidermal growth factor receptor distribution during chemotactic responses

Abstract

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

Figure 1

The EGFR–GFP chimera in E11 cells is functional. (A) Binding of 5 nM TMR–EGF on MTLn3 (n = 25) and E11 (n = 35) cells. Means and SEMs are plotted. Inset: Western blot showing the expression of the GFP construct in E11 cells, with the position of the 205-kDa molecular weight marker shown (arrow). (B) Correlation of EGFR–GFP expression (GFP fluorescence) with TMR–EGF binding (TMR fluorescence) on individual E11 cells. (C) Relative levels of EGF-induced lamellipod extension. Means and SEMs are plotted (n = 10 cells for each condition). (D) Chemotactic responses of E11 (squares, solid line) and MTLn3 (circles, dashed line) cells. Values given as mean and SEM of three different measurements.

Figure 2

The EGFR–GFP chimera is not concentrated at the leading edges of the extending lamellipods. (A) Distribution of the EGFR–GFP in unstimulated E11 cells. Left: Z-series images collected with a Noran confocal microscope as described in MATERIALS AND METHODS. Right: projection and corresponding transverse sections of the z-series shown on top. The crossed lines in the projection mark the positions at which the transverse sections were made. (B) Response of an E11 cell to the addition of 5 nM EGF at 0 s using GFP fluorescence. Thin arrows, ruffle; large arrows, extending lamellipod; arrowheads, large EGFR–GFP-containing vesicles. At right is a detail of a portion of the lamellipod of the same cell showing the formation of a group of small vesicles from a ruffle and the movement of the vesicles back from the leading edge over a total of 215 s. Bar, 10 um. (C) Quantitation of EGFR–GFP fluorescence in extending lamellipods. GFP fluorescence was quantitated as a function of distance from the leading edge of the lamellipod (0 μm). Mean ± SEM, n = 12.

Figure 2

The EGFR–GFP chimera is not concentrated at the leading edges of the extending lamellipods. (A) Distribution of the EGFR–GFP in unstimulated E11 cells. Left: Z-series images collected with a Noran confocal microscope as described in MATERIALS AND METHODS. Right: projection and corresponding transverse sections of the z-series shown on top. The crossed lines in the projection mark the positions at which the transverse sections were made. (B) Response of an E11 cell to the addition of 5 nM EGF at 0 s using GFP fluorescence. Thin arrows, ruffle; large arrows, extending lamellipod; arrowheads, large EGFR–GFP-containing vesicles. At right is a detail of a portion of the lamellipod of the same cell showing the formation of a group of small vesicles from a ruffle and the movement of the vesicles back from the leading edge over a total of 215 s. Bar, 10 um. (C) Quantitation of EGFR–GFP fluorescence in extending lamellipods. GFP fluorescence was quantitated as a function of distance from the leading edge of the lamellipod (0 μm). Mean ± SEM, n = 12.

Figure 2

The EGFR–GFP chimera is not concentrated at the leading edges of the extending lamellipods. (A) Distribution of the EGFR–GFP in unstimulated E11 cells. Left: Z-series images collected with a Noran confocal microscope as described in MATERIALS AND METHODS. Right: projection and corresponding transverse sections of the z-series shown on top. The crossed lines in the projection mark the positions at which the transverse sections were made. (B) Response of an E11 cell to the addition of 5 nM EGF at 0 s using GFP fluorescence. Thin arrows, ruffle; large arrows, extending lamellipod; arrowheads, large EGFR–GFP-containing vesicles. At right is a detail of a portion of the lamellipod of the same cell showing the formation of a group of small vesicles from a ruffle and the movement of the vesicles back from the leading edge over a total of 215 s. Bar, 10 um. (C) Quantitation of EGFR–GFP fluorescence in extending lamellipods. GFP fluorescence was quantitated as a function of distance from the leading edge of the lamellipod (0 μm). Mean ± SEM, n = 12.

Figure 3

Internalized EGFR–GFP accumulates on the source side of the cell in spatial gradients of EGF. A pipet containing 50 μM of EGF was placed next to an E11 cell at 0 s, and cell responses followed in phase (right) and with GFP fluorescence (left). Black arrows point to a ruffling area, showing EGFR internalization (white arrowheads). Bar, 10 um.

Figure 4

Movement of membrane vesicles during lamellipod extension. The cells were starved for 2 h then exposed to NBD-C6-SM for 30 min, followed by imaging on the Olympus CCD station. At time 0, 5 nM EGF with 0.35% BSA was added to the cells. The addition of BSA allows the visualization of recycling vesicles by removing the label from the plasma membrane. Phase contrast (A and B) and fluorescence (C and D) images of a cell 60 s (A and C) and 220 s (B and D) after the addition of EGF. (E) A superposition of the fluorescence images is shown together with the phase image of the top of the cell (B). It shows the paths taken for all the vesicles observed in the lamellipod area. The origin of each vesicle is shown as a white ring, and the different colors show the directions of movement of different vesicles (some of which split to form multiple progeny). The white outline indicates the position of the edge of the lamellipod (A) before extension has occurred. During EGF-induced lamellipod extension, most vesicles move toward the upper right side of the cell, although lamellipod extension occurs over the entire top of the cell. Bar, 10 um.

Figure 5

Transferrin receptors are not concentrated at the leading edges of extending lamellipods after EGF stimulation. Left: phase contrast image. Right, transferrin receptors visualized by immunofluorescence. Bar, 10 um.

Figure 6

EGF localization during chemotactic responses. (Above) TMR–EGF is concentrated on the surfaces of cells. A pipet filled with 250 nM TMR–EGF was placed near E11 cells, and a pressure pulse causing a saturating release of TMR–EGF was initiated at 0 s. Weak autofluorescence of the cells can be seen before the release of TMR–EGF. Phase (left) and TMR–EGF fluorescence (right) are shown. (Facing page) Internalized TMR–EGF and EGFR–GFP accumulate on the sides of cells closest to the micropipet. Pressure ejection from a pipet filled with 50 nM TMR–EGF was initiated at 0 s, and cells were imaged using phase contrast (left), GFP fluorescence (green, left), and TMR–EGF fluorescence (red, right). Overlap of green and red fluorescence generates a yellow color (far right image). Bar, 10 um..

Figure 6

EGF localization during chemotactic responses. (Above) TMR–EGF is concentrated on the surfaces of cells. A pipet filled with 250 nM TMR–EGF was placed near E11 cells, and a pressure pulse causing a saturating release of TMR–EGF was initiated at 0 s. Weak autofluorescence of the cells can be seen before the release of TMR–EGF. Phase (left) and TMR–EGF fluorescence (right) are shown. (Facing page) Internalized TMR–EGF and EGFR–GFP accumulate on the sides of cells closest to the micropipet. Pressure ejection from a pipet filled with 50 nM TMR–EGF was initiated at 0 s, and cells were imaged using phase contrast (left), GFP fluorescence (green, left), and TMR–EGF fluorescence (red, right). Overlap of green and red fluorescence generates a yellow color (far right image). Bar, 10 um..