Scaffold proteins are a hallmark of signaling pathways in eukaryotic cells. They are analogous to molecular circuit boards that wire the native signaling circuits which are composed of information-transferring enzymes like kinases and GTPases. IQGAP1 is a ubiquitously expressed scaffold that regulates cell state and morphology by tuning protein signaling pathways at the crux of phenotypic transitions in cancer biology and immunology. With > 6 domains, several proposed conformational states, and almost 2000 amino acids, IQGAP1 is known to bind and regulate actin, GTPases, MAP kinases, PI3K, E-cadherin, and numerous other proteins in diverse pathways. It has been shown theoretically and experimentally that scaffold proteins can activate or inhibit signaling pathways depending on their relative concentrations. Despite numerous experimental studies, very little is known about how living cells regulate scaffold protein concentrations, and how these ‘scaffolded’ protein complexes evolve dynamically. The Diehl lab has discovered novel endosomal compartments in epithelial cells, and these small compartments are enriched in IQGAP1, actin, phospholipids, and various membrane-binding proteins and are highly amenable to time-lapse microscopy for dynamical measurements. Using these compartments, we extracted micron-scale, multi-protein time series data with 60-second temporal resolution and used this data to resolve the extremely detailed coordination between IQGAP1, actin, membrane, and GTPases Cdc42 and Rac1, including how specific domains and residues of the scaffold contribute to its local concentration and compartment lifecycle-specific dynamics. By characterizing dynamics of mutant scaffolds, we discovered that IQGAP1 domains confer novel opposing behaviors: the GAP-related domain promotes IQGAP1 compartmental dissociation whereas the calponin homology domain limits its dissociation. We developed statistical models of compartmental protein dynamics to show how the dynamics of actin and the membrane can be predicted by observing a combination of wild type (WT) and mutant scaffold dynamics. While IQGAP1 is highly correlated with WT Rac1, we observed oscillatory dynamics between WT IQGAP1 and constitutively active Rac1, which suggests that there is a negative feedback loop involving IQGAP1 and the GTP-bound state of Rac1. This work presents the first detailed examination of the micron-scale dynamics of a scaffold protein with its signaling partners in living cells.