Chemoattractant-induced phosphatidylinositol 3,4,5-trisphosphate accumulation is spatially amplified and adapts, independent of the actin cytoskeleton

Abstract

Experiments in amoebae and neutrophils have shown that local accumulations of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] mediate the ability of cells to migrate during gradient sensing. To define the nature of this response, we subjected Dictyostelium discoideum cells to measurable temporal and spatial chemotactic inputs and analyzed the accumulation of PI(3,4,5)P(3) on the membrane, as well as the recruitment of the enzymes phosphoinositide 3-kinase and PTEN. In latrunculin-treated cells, spatial gradients elicited a PI(3,4,5)P(3) response only on the front portion of the cell where the response increased more steeply than the gradient and did not depend on its absolute concentration. Phosphoinositide 3-kinase bound to the membrane only at the front, although it was less sharply localized than PI(3,4,5)P(3). Membrane-bound PTEN was highest at the rear and varied inversely with receptor occupancy. The localization of PI(3,4,5)P(3) was enhanced further in untreated polarized cells containing an intact cytoskeleton. Interestingly, the treated cells could respond to two independent gradients simultaneously, demonstrating that a response at the front does not necessarily inhibit the back. Combinations of temporal and spatial stimuli provided evidence of an inhibitory process and showed that a gradient generates a persistent steady-state response independent of a previous history of exposure to chemoattractant. These results support a local excitation/global inhibition model and argue against other schemes proposed to explain directional sensing.

Fig. 1.

Measuring the input–output relationship. The spatial distribution of Cy3-cAMP concentration was quantified by measuring fluorescent intensity levels at points just outside the cell perimeter (red dots). Similarly, PI(3,4,5)P₃ levels were obtained by measuring the intensity of PH-GFP directly on the cell membrane (green dots) for both polarized (A) and latrunculin-treated (B) cells. Measured regions contained three adjacent pixels, and their averaged value was plotted. Only representative dots are shown, and at least 64 points were measured for each cell. At each point along the perimeter, where 0 denotes an arbitrary origin, the Cy3-cAMP (red) and PH-GFP (green) concentrations, normalized by their respective maxima, were plotted (Lower). (C)To quantify the degree of amplification, we plotted the output (PH-GFP concentration, normalized to the mean level for each cell) against the input (Cy3-cAMP concentration, normalized to its mean level). The dotted green line shows the expected plot for a system with no amplification. Data for three polarized cells (blue diamonds) and three latrunculin-treated cells (red squares) are included. Lines are least-squares fit.

Fig. 2.

Response to gradients of varying steepness and absolute concentrations. The input–output response of latrunculin-treated cells under varying chemotactic gradients was quantified as in Fig. 1. The micropipette location and pressure were altered to change the steepness and midpoint of the Cy3-cAMP gradient. Cells were exposed to steep gradients (needle near) and shallow gradients (needle far). The midpoint concentration of chemoattractant was varied by changing the pressure in the micropipette (pressure high and low). (B) Shown are the Cy3-cAMP and PH-GFP fluorescence levels for the same cell in two different gradients. Following the dotted line, it is clear that the same Cy3-cAMP concentration elicited vastly different PH-GFP responses. (C) Input–output data were normalized and graphed as in Fig. 1C for the four examples shown as well as eight other conditions in various cells. Here the responses coincided, showing that the cells' response depends on the relative gradient.

Fig. 3.

Response of PI 3′ enzymes. Input–output response of latrunculin-treated cells expressing PI3K-GFP (A) or PTEN-GFP (B) under varying chemotactic gradients was quantified as in Fig. 1. (A Upper) The pipette is located 10μm below the bottom-left corner of the frame; (B) the location is denoted by the asterisk. For both cell types, the individual responses coincided, showing that the enzyme responses also depend on the relative gradient. (C) To compare their relative degrees of amplification, we plotted input–output curves for the PI3K, PI(3,4,5)P₃, and the inverse of PTEN. Straight lines are least-squares fits.

Fig. 4.

Response to multiple simultaneous sources. Two micropipettes were brought in close proximity to latrunculin-treated cells creating cAMP profiles with two sharp gradients on either side. (A) PI3K-GFP localized to both ends of the cells. (B) PTEN-GFP relocalized to the cell membrane at the point of lowest cAMP concentration. (C) Cells expressing PH-GFP adjusted to changes in the Cy3-cAMP profile. At time 0 s, the concentration on the left was higher, whereas at 180 s, the profile was reversed. (D) pten–cells expressing PH-GFP respond to one micropipette at time 0 s. Note the broad crescent response on both cells. A second gradient was applied to the cell on the right at 30 s (not shown). A stable response is shown at 240 s. The cell on the right is incapable of responding with two sharp crescents as in C.

Fig. 5.

Response of cells to combinations of stimuli. Cells were exposed to sequential temporal and spatial stimuli, and images were captured. (A) A micropipette (location denoted by the asterisk) producing a stable Cy3-cAMP gradient was introduced to naïve cells after the first frame (0 s). (B) naïve cells (0 s) were stimulated by the addition of a micropipette producing a shallow chemoattractant gradient that was immediately pumped to generate a large transient stimulus. Fluorescent images of the Cy3-cAMP used in these experiments demonstrated that the stimulus from the initial bolus dissipated in the 4-ml chamber, and the stable gradient was established within 15 s (data not shown). (C) The previous experiment was repeated for cells originally in a gradient (0 s). The micropipette was pumped at 5 s. All results were reproducible (see Movies 1–3).

Fig. 6.

The LEGI model for gradient sensing. (A) Receptor occupancy regulates two opposing processes, excitation and inhibition, which together regulate the response (green, red, and black lines, respectively). When a cell is initially exposed to a gradient, both ends respond. The fast local excitation process increases proportionally to the local fraction of occupied receptors. The slow inhibitory response rises, driven by the global fraction of occupied receptors. When both processes reach a steady state (Lower), the profile of excitation along the length of the cell is proportional to the local fraction of receptor occupancy, whereas the global inhibitor is proportional to the mean level of occupied receptors. Thus, at the front of the cell, excitation exceeds inhibition, leading to a persistent response, whereas at the rear, inhibition exceeds excitation, and no positive response is elicited. (B) Our data suggest a model in which parallel LEGI mechanisms regulate PI3K and PTEN accumulations on the membrane. Their complementary action sharpens the PI(3,4,5)P₃ response.