RESUMO
Optogenetic modulation of protein interactions enables spatiotemporal control of cellular signaling dynamics in a variety of biological systems. However, light patterning by standard microscopes is limited by their complexity, sample throughput, and cost. To address the need for low-cost, user-friendly, and high-throughput photopatterning, we have engineered devices for light activation at variable amplitudes (LAVA). This protocol describes the assembly of LAVA devices, which enable spatial and temporal control of optogenetic stimulation and cellular signaling dynamics in multiwell cell culture plates. For complete details on the use and execution of this protocol, please refer to Repina et al. (2020).
Assuntos
Optogenética/instrumentação , Optogenética/métodos , Técnicas de Cultura de Células , Luz , Iluminação , Transdução de SinaisRESUMO
Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/ß-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.
