Heterotrimeric Gαβγ proteins are molecular switches that control eukaryotic signal transduction. In mammalian cell migration - a key process in immunity, inflammation, and development, - trimeric G proteins of the cAMP- inhibitory (Gi) family drive the movement of cells by transducing signals downstream of chemotactic G protein- coupled receptors such as the CXC chemokine receptor 4 (CXCR4). CXCR4 triggers heterotrimer dissociation at the plasma membrane, allowing βγ and GTP-bound Gαi to initiate their respective signaling cascades which, at the post-receptor level, are regulated and fine-tuned by numerous “accessory proteins”. Several hundred interactors have been described for the α-subunits of Gi proteins but their contribution to cell migration is not well understood. Best characterized chemotactic pathways are activated by Gβγ rather than Gαi, and for a long time, Gαi was considered but an instrument for timed release of Gβγ. Gαi is now increasingly appreciated as a signal transducer of its own, both via its cAMP-suppressing activity and via other, less studied effectors. The central aspect of Gαi signaling is that it must be spatially restricted and temporally coordinated between the different parts of the migrating cell. The intracellular gradients of Gαi activity and its ability to trigger both pro-migratory and anti-migratory signaling cascades on different time scales have been shown critical for directed cell motion. Unfortunately, the specific molecular interactions that maintain and interpret such asymmetric Gαi activity remain elusive, and so do the temporal aspects of their engagement. The objective of this proposal is to build a cohesive, spatiotemporally resolved picture of Gαi trafficking, activity, and regulation in live cells downstream of CXCR4. Through Aim 1 (Map the spatiotemporally resolved interactome of Gαi in CXCR4-expressing cells stimulated with CXCL12), we will determine when and where in the cell Gαi engages its known and so far undiscovered interactors in the course of CXCL12-triggered chemotactic signaling, with high spatial and temporal resolution, using the innovative approach of APEX2 proximity biotinylation. In Aim 2, we will fill an important methodological void by presenting a computational framework for converting APEX datasets for any desired target into systematic, large-scale, temporally resolved models of trafficking and regulation of that target in the signaling process of interest. By combining the Gαi APEX data of Aim 1 and tools of Aim 2, we will generate a model describing the trafficking and regulation of this key signal transducer in the process of CXCR4 migratory signaling. The model will help rationalize the observed phenotypic effects of perturbations, inform future experiments and, in the long run, assist target selection for novel combination therapies in inflammatory and other diseases. Altogether, this work will not only characterize Gαi trafficking and regulation in cell migration at an u...