Optotaxis: Light-Inducible Cytoskeletal Activators and the Automated Tracking of Directed Cell Migration

Cell migration in response to a stimulus is a complex and highly regulated whole-cell process. Detection of subtle phenotypic migration changes has been difficult at a single-cell level, necessitating the use of mass migration assays with many dozens or hundreds of individual cells migrating over the course of hours or days. However, the lack of automated tools has made this analysis highly tedious and prone to human bias. Additionally, cell migration can be directed by gradients of extracellular cues as intracellular cytoskeletal networks and regulatory pathways convert those signals into mechanical forces. Most migration assays provide stimulus in the form of a diffusible chemical gradient; however, such gradients are complex to maintain over the timescales necessary to monitor directed migration and are difficult to interpret mechanistically as single receptors activate parallel downstream pathways that become difficult to disentangle. In this work, we constructed an automated pipeline for the segmentation, tracking, and migration analysis of migrating fibroblasts visualized via low magnification widefield microscopy. Using optogenetically activated intracellular signaling proteins, such as the Rac-activating Tiam1, and projecting a tunable gradient of (488 nm) light onto the sample, we stimulated cellular migration - or “optotaxis” - in a consistent and measurable manner. 24 hour time-lapses were analyzed via the automated pipeline, and significant optotaxis towards brighter regions was observed consistent with hand-tracking analysis. Overall, these efforts establish the strong foundation of an adaptable high-throughput system for studying the internal signaling involved in directed cell migration