UC San Diego

All biological processes arise through the coordinated actions of biochemical pathways. How such functional diversity is achieved by a finite cast of molecular players remains a central mystery in biology. Spatial compartmentation-the idea that biochemical activities are organized around discrete spatial domains within cells-was first proposed nearly 40 years ago and has become firmly rooted in our understanding of how biochemical pathways are regulated to ensure specificity. However, directly interrogating spatial compartmentation and its mechanistic origins has only really become possible in the last 20 or so years, following technological advances such as the development of genetically encoded fluorescent biosensors. These powerful molecular tools permit a direct, real-time visualization of dynamic biochemical processes in native biological contexts, and they are essential for probing the spatial regulation of biochemical activities. In this Account, we review our labs efforts in developing and using biosensors to map the spatial compartmentation of intracellular signaling pathways and illuminate key mechanisms that establish the boundaries of an intricate biochemical activity architecture. We first discuss the role of regulatory fences, wherein the dynamic activation and deactivation of diffusible messengers produce diverse signaling compartments. For example, we used biosensors for the Ca2+ effector calmodulin and its downstream target calcineurin to reveal a spatial gradient of calmodulin that controls the temporal dynamics of calcineurin signaling. Our studies using cyclic adenosine monophosphate (cAMP) biosensors have similarly elucidated fenced cAMP domains generated by competing production and degradation pathways, ranging in size from cell-spanning gradients to nanoscale hotspots. Second, we describe the role played by intracellular membranes in creating unique signaling platforms with distinctive pathway regulation, as revealed through studies using subcellularly targeted fluorescent biosensors. Using biosensors to visualize subcellular extracellular response kinase (ERK) pathway activity, for example, led us to discover a local signaling circuit that mediates distinct plasma membrane ERK dynamics versus global ERK signaling. Similarly, our work developing biosensors to monitor the subcellular mechanistic target of rapamycin complex 1 (mTORC1) signaling allowed us to not only clarify the presence of mTORC1 activity in the nucleus but also identify a novel mechanism governing the activation of mTORC1 in this location. Finally, we detail how molecular assemblies enable the precise spatial tuning of biochemical activity, through investigations enabled by cutting-edge advances in biosensor design. We recently identified liquid-liquid phase separation as a major factor in cAMP compartmentation aided by a new strategy for targeting biosensors to endogenously expressed proteins via genome editing, for instance, and have also been able to directly visualize nanometer-scale protein kinase signalosomes using an entirely new class of biosensors specifically developed for the dynamic super-resolution imaging of live-cell biochemical activities. Our work provides key insights into the molecular logic of spatially regulated signaling and lays the foundation for a broader exploration of biochemical activity architectures across multiple spatial scales.