Genetically Encoded Multimeric Nanoparticles for Measuring Intracellular Diffusion and Cytoplasmic Viscosity

The crowded cytoplasmic environment affects the transport and reactions of macromolecular complexes at the mesoscale (10-100 nm). Cytoplasmic viscosity correlates with, or even controls, cellular physiology and cellular states. Quantitative measurement of intracellular diffusion is therefore important. Such measurements require inert probes of defined size that do not perturb the cellular environment. Genetically Encoded Multimeric Nanoparticles (GEMs) meet these requirements. GEMs are self-assembling fluorescent protein nanocages. A scaffold protein fused to a fluorescent protein assembles into a bright, stable particle of defined geometry. GEMs are genetically encoded and require no microinjection. Their multimeric assembly makes the fluorescent particles bright enough for rapid single-particle tracking. This chapter provides a protocol for measuring cytoplasmic diffusion using 40 nm GEMs. The workflow covers cell preparation, live-cell imaging, single-particle tracking, and mean-squared-displacement analysis. The Notes section addresses theoretical background, parameter optimization, statistical design, and alternative configurations.