The role of dynamics in defining the limits of normal developmental signaling

总结

Summary In this supplement, we request funding to purchase a live-cell fluorescence microscope that will replace failing essential equipment, and which will significantly upgrade our ability to collect cell signaling biosensor data with high spatial and temporal resolution. The parent project for this supplement is centered on using live-cell biosensor experiments to collect kinetic data on kinase activity and gene expression. These datasets uniquely enable computational modeling of the relationship between signal transduction pathways and gene expression. Because of the time-intensive nature of these long-term time-lapse imaging experiments, we need a dedicated live-cell microscopy system that can run continuously for multi-day experiments. Our current systems have enabled significant conceptual and methodological advances in growth factor signaling but face two major problems: 1) frequent down-time due to failures of aging equipment and 2) outdated technology that limits our ability to collect high-quality data from live cells. The proposed instrument would dramatically decrease the amount of personnel time lost to component failures. Furthermore, the proposed instrument will take advantage of a large field of view sCMOS camera that can collect data from nearly twice as many cells in each experiment; these additional data will directly improve our computational modeling efforts by capturing more unique cellular behaviors. Finally, the new instrument will include automation to enable the use of 40X and 60X water immersion objective lenses for long-term time time-lapse experiments. Relative to our current imaging systems that rely on 20X and 40X non-immersion lenses, these lenses will increase spatial resolution and sensitivity, enabling experiments in which we can link signal transduction and gene expression to morphological changes in key organelles such as autophagosomes and mitochondria, which our work has implicated in regulation of the dynamic kinase activity being studied.