![]() The effect of crowding on particle motion is particularly relevant for large, multinucleate cells, where distinct functional territories emerge within the cytosol. These impacts of cytosolic crowding are not limited to eukaryotes but have also been seen in bacterial cells ( Parry et al., 2014 Gray et al., 2019). Additionally, it has been shown that glucose starvation can lead to a drastic reduction in mobility of molecules in the cytosol, indicating that cells may be able to tune their cytosolic material properties as a survival mechanism ( Joyner et al., 2016 Munder et al., 2016). Conversely, some reactions may be potentiated by crowding through entropic depletion attraction effects, which favor molecular interactions ( Zhou et al., 2008) and molecular condensation ( Delarue et al., 2018). For example, the diffusion of signaling molecules could be much slower within crowded regions of the cytosol delaying the downstream response in those regions. Crowding may therefore have a critical influence on cell function, as molecular motion directly affects a broad range of physiological processes ( Bressloff and Newby, 2013). Molecular crowding can alter the material properties of the cytosol, which in turn influences the thermal, entropic fluctuations of molecular species within, such as Brownian motion of small molecules. Thus, the structure of the cytosol across length scales and the effect that this has on biochemistry are still a highly debated topic in cell biology. It is not well understood to what degree the motion of macromolecular complexes on the scale of ∼10–100 nm is impacted by crowding, or how crowding may impact the formation of biomolecular condensates within cells. Measurements of colloid osmotic pressure have been used to argue for a less crowded picture of the cytosol at the length scale of single proteins (<10 nm) due to the potential for many macromolecules to exist in higher-order complexes ( Mitchison, 2019). Furthermore, substantial molecular crowding within the cytosol has been predicted due to the high concentration of macromolecules. With electron microscopy, it became clear that the cytoskeleton, endomembrane systems, and protein translation machinery can generate a crowded landscape in the cytosol. Wilson expanded on the possible explanations for the meshwork appearance of the cytosol and described it as an emulsion in which multiple liquids of different chemical and physical properties coexist ( Wilson, 1899). The nature of the cytosol has been speculated about since the first glimpses of cells in primitive microscopes, yet the cytosolic environment experienced by macromolecules across different length scales remains elusive today. ![]() ![]() Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20–60 nm). Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. The spatial structure and physical properties of the cytosol are not well understood.
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