Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

Abstract

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96‐well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 si RNA perturbations. A key finding was that the G‐protein G i α subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP . The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low‐ and high‐resolution fluorescence microscopy. image A new strategy, involving optical shaping of gradients, allows systematically analyzing components regulating cell migration speed and directionality. The approach is applied to characterize migration and chemotaxis phenotypes for 285 si RNA perturbations in human neutrophils. Automated uncaging of attractants allows systematic live‐cell imaging of chemotaxis. Leukocytes have distinct components specialized for regulating cell speed and cell direction in response to chemoattractant gradients. Specialization in the chemoattractant signaling pathway occurs already at the level of the G‐proteins.