3'-phosphoinositides regulate the coordination of speed and accuracy during chemotaxis

Abstract

The PI3K/PTEN pathway, as the regulator of 3'-phosphoinositide (3'-PI) dynamics, has emerged as a key regulator of chemoattractant gradient sensing during chemotaxis in Dictyostelium and other cell types. Previous results have shown 3'-PIs to be important for regulating basal cell motility and sensing the direction and strength of the chemoattractant gradient. We examined the chemotaxis of wild-type cells and cells lacking PTEN or PI3K1 and 2 using analytical methods that allowed us to quantitatively discern differences between the genotype's ability to sense and efficiently respond to changes in gradient steepness during chemotaxis. We found that cells are capable of increasing their chemotactic accuracy and speed as they approach a micropipette in a manner that is dependent on the increasing strength of the concentration gradient and 3'-PI signaling. Further, our data show that 3'-PI signaling affects a cell's ability to coordinate speed and direction to increase chemotactic efficiency. Using to our knowledge a new measurement of chemotactic efficiency that reveals the degree of coordination between speed and accuracy, we found that cells also have the capacity to increase their chemotactic efficiency as they approach the micropipette. Like directional accuracy and speed, the increase in chemotactic efficiency of cells with increased gradient strength is sensitive to 3'-PI dysregulation. Our evidence suggests that receptor-driven 3'-PI signaling regulates the ability of a cell to capitalize on stronger directional inputs and minimize the effects of inaccurate turns to increase chemotactic efficiency.

FIGURE 1

Experimental and analytical paradigm to measure directed and persistent random motility. (A) Quantitation of fluorescein (MW 332.3) diffusing from a micropipette reveals the nonlinear concentration profile in the assay. (B) Schematic of the microfluidic device used in the study; manipulation of the flow into the two inlets (labeled cAMP and buffer) enables the production of stable concentration gradients. Inset, image of fluorescein fluorescence in the device. (C) Quantification from a line scan of fluorescein image in B detailing experimental control over chemoattractant profiles. Flowing buffer and cAMP at identical flow rates creates linear chemoattractant concentration gradients for chemotaxis that are stable over experimental timescales (<90 min). (D) Time series showing the chemotaxis of wt cells in the microfluidic device when exposed to a linear cAMP gradient of 50 pM/μm cAMP with a midpoint of 12.5 nM (concentration increases from right to left and flow is from top to bottom). For clarity, five cells were chosen and their respective tracks are shown in white.

FIGURE 2

Chemotactic accuracy and speed in linear and nonlinear concentration gradients. The chemotactic accuracy (A) and speed (D) of the three genotypes versus distance from the highest concentration in the nonlinear gradient (left) and linear gradient (right). Mean accuracy (B) and speed (E) for wt, pi3k1/2-, and pten- in the micropipette (nonlinear) and microfluidic (linear) assay, respectively. Statistical comparison of the rate of improvement of accuracy (C) and speed (F) in the micropipette assay and the linear region of the microfluidic device. For comparison, the rate of increase in each chemotactic parameter was normalized to the rate of wt cells in the micropipette assay. Stars represent statistically significant differences at p < 0.05.

FIGURE 3

During chemotaxis, accurate movements tend to be faster movements. (A) Two-dimensional histograms displaying the log of the probability that a cell will make a movement during chemotaxis in the micropipette and microfluidic assay with a particular angle (x axis) and speed (y axis) represented in grayscale. Speed values are separated in bins 5 μm/min wide; angles are in bins 15° wide and each bin is labeled with the upper limit for that bin. The heat-mapped insets show the difference between a given mutant and wt. Doubling and halving the bin widths had no appreciable effect on the general conclusion. Similar patterns can be seen in 2-D histograms of movements from the microfluidic device. (B) Scatter plot of the accuracy of wt movements versus their speed after normalized to each individual cell's mean speed. A linear fit (red line) indicates the correlation between accuracy and speed. (C) Correlation coefficients between each genotype's accuracy and normalized speed (as in B). Each correlation was found to be significant at p < 0.05 level. (D) A magnification of the plot from B. This plot, containing >99% of the total data, can be divided into four regions (labeled I, II, III, and IV) that describe the accuracy and speed of the cell (see text). (E) Percent of movements from each genotype with positive accuracy (quadrants III and IV in D). (F) Percent of accurate and inaccurate movements with faster than average speeds.

FIGURE 4

Simulations of coordination reveal its effect on chemotactic efficiency. (A) “Tracks” of cells from a stochastic simulation of chemotaxis with varying degrees of coordination between directional accuracy and speed. (B and C) The probability distribution of the speed and direction, respectively, of cells in A. (D) The two-dimensional probability distributions reveal the dependency of directional accuracy and speed in the simulations in A. (E and F) Mean speed and direction for each simulation in A. (G) Mean coordination index for each simulation. The stars indicate differences at the p < 0.05 level by Bonferroni t-test after ANOVA.

FIGURE 5

Coordination in linear and nonlinear concentration gradients. (A) Coordination versus distance from the highest chemoattractant concentration present in the nonlinear gradient (left) and linear gradient (right). (B) Comparison of the mean coordination of the three genotypes in the linear and nonlinear gradients. (C) Statistical comparison of the rate of improvement of coordination in the micropipette assay and the linear region of the microfluidic device. For comparison of the increase in the coordination, the rate of increase was normalized to the rate of wt cells in the micropipette assay. Stars represent statistically significant differences at p < 0.05.