Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion Studies

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Abstract Single particle tracking (SPT) is a powerful technique for probing the diverse physical properties of the cytoplasm. Genetically encoded nanoparticles provide an especially convenient tool for such investigations, as they can be expressed and tracked in cells via fluorescence. Among these, 40-nm GEMs provide a unique opportunity to explore the cytoplasm. Their size corresponds to that of ribosomes and big protein complexes, allowing us to investigate the effects of the cytoplasm on the diffusivity of these objects while excluding the influence of chemical interactions during stressful events and pathological conditions. However, it has been shown that cytoplasmic viscosity is tightly regulated and plays a crucial role in maintaining homeostasis during protein synthesis and degradation. Despite this, the effects of GEM expression levels on diffusivity remain largely uncharacterized in mammalian cells. To optimize the GEMs tracking and estimate GEMs-expression effects we constructed dox-inducible GEM expression system and compare with a previously reported constitutive expression system. The optimized level of GEMs expression increases the measured diffusivity from 0.29 ± 0.02 μm 2 /sec in GEMs-overexpressed cells to 0.35 ± 0.02 μm 2 /sec; improve homogeneity throughout the cell population; and facilitates particle tracking. We also improved the analyses of GEM diffusivity by applying effective diffusion coefficient while considering the type of motion and assessing the heterogeneity in the type of motion by calculating the standard deviations of particle displacements. Statement of significance Describing cytoplasmic properties, such as environmental viscosity and protein complex motion, is essential for understanding molecular-level changes in cell function and pathology. A recently developed approach uses self-assembling fluorescent protein probes, expressed in cells, to investigate cytoplasmic properties through single-particle tracking (SPT). One such system employs genetically encoded multimeric (GEM) nanoparticles— scaffold protein structures similar in size to ribosomes. This study addresses a key limitation in SPT of GEMs by examining how varying GEM expression levels affect measured diffusivity and tracking quality in mammalian cells. Our findings demonstrate that controlled GEM expression reduces particle overcrowding, increases measured diffusivity, and enhances track detection. This work contributes valuable insights into optimizing GEM nanoparticle applications for studying cytoplasmic viscosity and motion dynamics.

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