Synthetic spatially graded Rac activation drives cell polarization and movement

Abstract

Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.

Fig. 1.

Graded activation of Rac directs cellular polarity. (A) Schematic of the mechanism of Rac activation by rapamycin-induced heterodimerization. (B) Microfluidic device used to generate linear gradients of rapamycin with a sample image of the microchannels seeded with individual HeLa cells and the corresponding gradient visualized with Alexa 594 dye. Ports are labeled according to function. The red layer of the device is the fluid flow layer, and the green layer is the control valve layer. Alexa 594 dye is used to visualize the gradient (red) in all subsequent images. A sample image of a cell transfected with the Rac activator, YF-TIAM1, experiencing a gradient of rapamycin is shown below. (C–F) Four polarity states observed after the attachment period with respect to the direction of the imposed rapamycin gradient and associated polarization responses to the gradient of rapamycin. Images are rotated by 90° to aid in visualization. Cartoons illustrate the polarity of the associated state and the direction of the gradient. The green color indicates expression of YF-TIAM1. Yellow arrowheads denote the initial direction of polarity. The rapamycin gradient is shown in the 5-min image and removed in subsequent images for clarity. Red dotted lines highlight evolving changes in cell morphology. Times are given in minutes. (Scale bars, 10 μm.) (G–J) Kymographs taken across cell centers (specified in accompanying image) illustrate the morphological changes of the corresponding cells in C–F over the experimental period. Blue lines trace initially polarized faces, and red lines trace initially unpolarized faces. Yellow arrowheads denote the initial response time, and their location indicates which cell face was the first to change in the gradient. White arrowheads indicate the late polarization time. Times are given in minutes.

Fig. 2.

Analysis of the initial response time. (A) Sample kymograph of the mathematical model simulation under graded Rac stimulation. (B) Model response time vs. normalized gradient (s₁) for two different values of s_o (input). (C–E) Kymographs chronicle typical changes in cell morphology seen during early time periods. The first image before each kymograph depicts the gradient (red) that the cell is experiencing as visualized with Alexa 594 dye. The gradient is not shown in resulting images to promote clarity of morphological changes. The green color indicates expression of YF-TIAM1. Red dotted lines highlight evolving cell boundaries. Yellow arrowheads indicate initial polarities. Times are given in minutes. (Scale bars, 10 μm.) (F) Dependence of initial response times on gradient values. States are color-coded: state I, green (n = 29); state II, blue (n = 37); and state III, red (n = 27). In each plot, the colored dots highlight the dependence of that particular state and the gray dots illustrate where the response times of the other states fall. Data are fitted based on simulation results. Spearman correlation coefficient: state I = −0.679, state II = −0.655, and state III = −0.583. Τhe asterisk indicates a statistically significant difference (state II vs. state I, P < 1e-4; state II vs. state III, P < 1e-3) between the state II curve vs. the other states. There is no statistical difference between state I and state III (P = 0.54). Both tests were carried out using an F test.

Fig. 3.

Analysis of the late response time. (A) Kymograph depicts the change in the lamellipodium directed by the gradient and dimming seen in the cell body during the late polarization time. The image preceding the kymograph illustrates the gradient (red), visualized with Alexa 594, received by the cell. The green color visualizes expression of YF-TIAM1. Red dotted lines indicate where the fluorescent values in C are taken from. Times are given in minutes. (Scale bar, 10 μm.) (B) Simulations show response strength vs. input (s_o) for two gradient levels (s₁); note bifurcations at distinct s_o values. Response strength is defined as the ratio of Rac activity at the front vs. the back in the model cell. (C) Intensity of the cell body normalized to the initial time point. Intensity values are taken as the mean of the fluorescence intensity of the area enclosed by the red trace in A. The drop line indicates the late polarization time, with a circle highlighting the inversion of response used to define this time. (D) Three-dimensional reconstruction of confocal slices of the same cell taken before and after rapamycin addition. The “post” cell image was taken 240 min after treatment. (Scale bars, 10 μm.) (E) Cell body volume before and after rapamycin addition. The data show the mean of n = 9 cells, and error bars show the SEM. The asterisk denotes a statistically significant difference (P = 0.019), using a two-sided Student’s t test. (F) Late response time as a function of mean concentration: state I, green (n = 29); state II, blue (n = 37); and state III, red (n = 27). The response times of other states are superimposed on each plot in gray. The pink drop line in the state III plot demarcates the separation point between unresponsive cells and responsive cells. Pearson correlation coefficient of linear regressions: state I = −0.608, state II = −0.783, and state III = −0.698. The red asterisk denotes a statistically significant difference (state II vs. state I, P < 1e-4; state II vs. state III, P < 1e-4) between the y intercept of the linear regression for state II vs. the y intercepts of the regression data from other states. There is no significant difference between the y intercept of state I vs. state III (P = 0.13). Both statistical analyses were carried out using an ANCOVA test.

Fig. 4.

Overcoming a Rac activity threshold determines the late polarization time. (A and D) Time series of representative cells stimulated with a gradient of rapamycin for 30 min and 60 min, respectively. Yellow arrowheads indicate the initial direction of cell polarity. Times are given in minutes. (Scale bars, 10 μm.) (B and E) Kymographs taken from the center of cells (specified in accompanying image) illustrate morphological changes seen in A and D, respectively. Blue lines track the initially polarized cell face, and red lines track the opposite face. Below the kymograph is the experimental scheme used to stimulate the cell. (C and F) Late polarization time for 30-min and 60-min stimulation periods for all three states, respectively. For 30 min: state I, green (n = 17); state II, blue (n = 13); and state III, red (n = 31). For 60 min: state I, green (n = 32); state II, blue (n = 37); and state III, red (n = 32). Diamonds indicate cells that did not respond within the experimental time frame (240 min), and circles indicate responsive cells. Gray dots in each plot represent the late polarization times for cells in each state from Fig. 3F. The drop lines demarcate a threshold between responding and nonresponding cells.

Fig. 5.

PI3K modulates Rac-mediated polarization. (A) Late polarization time dependence against mean concentration with LY294002 treatment. State I, green (n = 21); state II, blue (n = 31); and state III, red (n = 27). Diamonds represent nonresponder cells, and circles represent responding cells. Gray dots on each plot illustrate the late polarization times seen in Fig. 3F. The gray drop line in the state III cell response plot represents the previous response threshold between nonresponding and responding untreated cells, and the colored line represents the shifted threshold following LY294002 treatment. (B) Model schematic with the red “X” indicating that feedback from PIP₃ (f₁) is decreased during the subsequent simulations. (C) Simulations of response strength vs. signal strength for different feedback levels (in all previous simulations, f₁ = 1). (D) Response threshold for state III cells with and without LY294002 treatment, respectively. The drop lines indicate the response threshold.

Fig. P1.

Externally applied gradients of a Rac activator drive cell polarization and motility. (A) In metastasis, cancer cells exhibit directed migration toward external gradients of chemoattractants. Chemoattractants bind and activate receptors on the cell surface, which, in turn, localize guanine exchange factors (GEFs) and lipid kinases, such as PI3K, to their base. GEFs activate Rho GTPases, such as Rac, which induce the formation of protrusions. Protrusions are localized to bound receptors and lead to migration toward chemoattractants. (B) Gradient of Rac activator induces directed migration. Cells are initially distributed randomly into different polarity states. Rapamycin enters through the cell surface and directly localizes a Rac GEF to the membrane, causing initial morphological changes driven by changes in the distribution of Rac activity. The magnitude of the applied gradient regulates the timing of these initial changes. Continued exposure to the gradient causes polarization and motility. The time necessary to polarize is dependent on the mean value of the gradient and feedback from PI3K. Synthetic directed migration can recreate features of physiological directed migration.