Self-assembled nanoparticles give insight into the regulation of macromolecular crowding.
The interior of a cell is packed with biomolecules, and this crowding has important effects on the proper function of living systems. For example, increased crowding can enhance the association of molecules, and can also affect their diffusion kinetics. Thus, it is not surprising that cells regulate the amount of macromolecular crowding in order to finely control intracellular reactions.
The study of the mechanical properties of a cell is known as microrheology. Many microrheological methods involve injecting a probe into a cell and monitoring its motion by microscopy. From these measurements, one can determine features such as the viscosity and elasticity of the milieu, and even map the regions of a cell that are accessible to the probe. However, microinjection can be technically challenging and limits the types of samples that can be observed. To bypass these challenges, Benjamin Engel at the Max Planck Institute of Biochemistry, Liam Holt at New York University, and their research groups developed genetically encoded multimeric nanoparticles (GEMs) as microrheology probes.
The GEMs are composed of scaffolding domains based on archaeal and bacterial proteins fused to a fluorescent protein, which self-assemble into brightly fluorescent 20- and 40-nm particles, respectively, when expressed in cells. These GEMs are roughly the same size as large molecular complexes such as the ribosome, and are therefore useful probes for studying the environment encountered by such complexes.
After characterizing the GEMs with several approaches, including electron microscopy and cryoelectron tomography, and demonstrating that they form spontaneously in yeast and mammalian cells, the researchers used them to probe the regulation of intracellular crowding by mTORC1, the major sensor of amino acids in eukaryotes. By altering growth conditions, applying mTORC1 inhibitors, and using mTORC1 mutants while examining microrheology using the GEMs, they discovered that mTORC1 controls macromolecular crowding by regulating the concentration of ribosomes in the cytoplasm.
Delarue, M. et al. mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell 174, 338–349 (2018).
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Strack, R. Probes for molecular crowding. Nat Methods 15, 570 (2018). https://doi.org/10.1038/s41592-018-0098-8