Probing Directed Cell Migration By Synthetically Generating Intracellular Gradient of Signaling Molecules

Polarized cells possess intracellular gradients of active Rho GTPases which serve to properly localize and direct cytoskeletal machinery for optimal motility. The pathways regulating the distribution and activity of the Rho GTPases are still being unraveled and at a fundamental level, it is unknown whether intracellular gradients of the Rho GTPases themselves are sufficient to drive polarity and motility (Movie #2). This is an interesting question, especially because we have previously shown that the neutrophils can polarize with uniform PI3K activation (Movie #3), but not with that of Rac. Thus, we have combined two technologies, chemically-inducible molecular probes and microfluidics, to present cells with a novel perturbation- an induced linear gradient of active, endogenous Rac without receptor actuation. We found that the chemical inducer gradient was sufficient to direct cells towards the chemical source, regardless of their initial direction of polarization or lack thereof (Movie #4). We created a simplified mathematical model of a spatial network of Rho GTPases to model this perturbation and found that the model was fully predictive of the data observed. Consistent with the model prediction, we found that the initial chemotactic response time was primarily dependent upon the steepness of the Rac activity gradient but not the concentration of the active Rac. Our joint microfluidics and chemical activation methodology to direct cells with shallow spatial gradients has great potential utility in further studies of cell polarity and motility.  We are now expanding the studies to other Rho GTPases as well as their upstream regulators to systematically map out core components in directed cell migration.
Graded activation of Rac with microfluidcs and rapamycin directs cellular polarity. (Lin et al. PNAS, 2012)