The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. Multi-domain scaffold proteins orchestrate the activities of multiple signaling intermediates and regulatory proteins on organelles. Investigating scaffold functions is challenging because these interactions occur at different timescales that are not well understood through standard static colocalization analyses. This work employs live-cell imaging to probe how the multi-domain scaffold IQGAP1 coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling, and point to a scaffold tethering mechanism. This work also focuses on engineering mammalian cells to modulate steady-state protein expression. Protein abundance is a highly regulated cellular process, and variations in protein levels leads to changes in cellular phenotypes and functions. These changes may cause human genetic diseases and changes in immune response, and it is important to build tunable mammalian cell libraries that can be used for systematic analyses to study the link between protein expression levels and cellular function. In this project, mammalian cell libraries with subpopulations of variable steady-state expression of a fluorescent protein or membrane ligand were made by multiplexing synthetic biology strategies in a modular, plug-and-play, high-throughput system. These libraries generated cell lines with tunable, stable, robust expression with quick turnaround times, which can be employed for future functional analyses of protein expression levels on cellular activity.