| Control of cell migration and directional persistence underlie many important physiological processes, from wound healing to metastasis. Mesenchymal cells, such as skin fibroblasts, migrate in a manner that is characterized by multiple competing protrusions, weak polarization, and strong adhesive forces. External cues such as chemical gradients and substrate stiffness work in concert with internal signaling events to direct cell movement. Efficient reorganization of the actin cytoskeleton in response to those signals is critical; however, the manner in which cells interpret internal and external cues and translate them to bias migration is poorly understood.;The lipid kinase phosphoinositide 3-kinase (PI3K) has well known roles in cell proliferation and survival and has been broadly implicated in cell migration. However, its specific role in the context of mesenchymal migration has yet to be elucidated. In this work, we describe a PI3K-dependent mechanism by which mesenchymal cells achieve large-scale reorientation of migration. We found that they do so by forming branched lamellipodia; if extension of the lamellipodia propagates to the fullest extent, the cell performs a ninety-degree turn. Inhibition of PI3K did not significantly reduce the instantaneous speed of migration or the frequency of branch formation, rather it abrogated the stability and propagation of the branched state. Thus, a PI3K-inhibited cell typically exhibits an elongated morphology and migration biased along its long axis. Dynamic recruitment of PI3K activity apparently responds to protrusion, and accordingly we found that it can be induced via actin polymerization spurred by photo-activation of the small GTPase, Rac. Conversely, inhibition of actin polymerization prevents protrusion and dynamic relocalization of PI3K. In cell migration directed by an external gradient (PDGF chemotaxis), the branch that is exposed to the highest concentration of chemoattractant is favored, resulting in alignment of the cell upgradient.;Having established a critical role for PI3K and lamellipodial branching in mesenchymal migration, we sought to discover the mechanisms by which new branches were formed and how PI3K activity originated in these structures. Analysis of high-resolution migration movies revealed that finger-like actin bundles called filopodia often precede and direct the formation of new lamellipodia. Using quantitative image analysis protocols that we developed, we showed that nearly all lamellipodia are biased by filopodia or submembranous actin bundles. To test this further, we depleted as well as overexpressed the actin-bundling protein, fascin-1, in different populations of cells and quantified the number of morphological extensions. As expected, the number of cellular extensions varied according to the fascin levels. Based on the finding that actin bundles template lamellipodial protrusion, we hypothesized that they also prime the recruitment of PI3K signaling. Using ratiometric imaging, we found that PI3K is indeed enriched within filopodia. By imaging focal adhesions and perturbing cells with pharmacological inhibitors, we found that adhesions serve as hubs for PI3K signaling primarily through the recruitment of focal adhesion kinase (FAK). This adhesion-based pathway fosters actin polymerization, dilation of lamellipodia, and the formation of new adhesions, thus amplifying signaling; under some conditions, this process manifests as periodic protrusion waves. Depletion of fascin ablated direction migration in response to a fibronectin gradient (haptotaxis) but not PDGF chemotaxis. As fibroblast filopodia contain primed integrins at their tips which bind specifically to fibronectin, this suggests a possible role for filopodia in haptotactic sensing; where increased ligand density promotes the binding of filopodia and therefore, the eventual direction of migration.;To obtain the quantitative data in the studies mentioned above, a host of image analysis methods were developed. Here we present some of the techniques we developed for analysis of florescence, differential interference contrast (DIC), and phase contrast images. These techniques primarily involve the segmentation of cytoskeletal or signaling structures/domains for the purpose of tracking, comparison, or quantification. We demonstrate analyses that utilize signals from particular fluorescent probes and methods that identify subcellular structures based on morphological criteria, these structures can be analyzed directly or masks of these structures can be used to spatiotemporally restrict subsequent signaling analyses. These techniques offer quantitative approaches for assessing signaling and cytoskeletal phenomena in migrating cells. |
