Visualizing in Vivo Dynamics of Designer Nanoscaffolds

Enzymes of natural biochemical pathways are routinely subcellularly organized in space and time in order to improve pathway efficacy and control. Designer scaffolding platforms are under development to confer similar benefits upon engineered pathways. Herein, we evaluate bacterial microcompartment shell (pfam0936-domain) proteins as modules for constructing well-defined nanometer scale scaffolds in vivo. We use a suite of visualization techniques to evaluate scaffold assembly and dynamics. We demonstrate recruitment of target cargo molecules onto assembled scaffolds by appending reciprocally interacting adaptor domains. These interactions can be refined by fine-tuning the scaffold expression level. Real-time observation of this system reveals a nucleation-limited step where multiple scaffolds initially form within a cell. Over time, nucleated scaffolds reorganize into a single intracellular assembly, likely due to interscaffold competition for protein subunits. Our results suggest design considerations for using self-assembling proteins as building blocks to construct nanoscaffolds, while also providing a platform to visualize scaffold-cargo dynamics in vivo.