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Cytoplasmic organization promotes protein diffusion in Xenopus extracts

Huang WYC, Cheng X, Ferrell JE.

Nat Commun. 2022 Sep 23;13(1):5599. doi: 10.1038/s41467-022-33339-0.


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The cytoplasm is highly organized. However, the extent to which this organization influences the dynamics of cytoplasmic proteins is not well understood. Here, we use Xenopus laevis egg extracts as a model system to study diffusion dynamics in organized versus disorganized cytoplasm. Such extracts are initially homogenized and disorganized, and self-organize into cell-like units over the course of tens of minutes. Using fluorescence correlation spectroscopy, we observe that as the cytoplasm organizes, protein diffusion speeds up by about a factor of two over a length scale of a few hundred nanometers, eventually approaching the diffusion time measured in organelle-depleted cytosol. Even though the ordered cytoplasm contained organelles and cytoskeletal elements that might interfere with diffusion, the convergence of protein diffusion in the cytoplasm toward that in organelle-depleted cytosol suggests that subcellular organization maximizes protein diffusivity. The effect of organization on diffusion varies with molecular size, with the effects being largest for protein-sized molecules, and with the time scale of the measurement. These results show that cytoplasmic organization promotes the efficient diffusion of protein molecules in a densely packed environment.


Figure 1. a Schematic of the experimental setup used in this study: Xenopus laevis eggs were fractionated to obtain the undiluted cytoplasmic extract, which was then deposited on the surface of an imaging dish and covered with mineral oil. b Time-lapse images of the self-organizing cytoplasm, visualized with 1 µM ER-Tracker Red dye. c Image (left) of dilute 100-nm (diameter) microspheres in extracts. Trajectories (right) are examples from single-particle tracking in the organized cytoplasm (interior regions in Fig. 2a). The relative positions between the three tracked particles were adjusted for display (they were tens of microns apart in the extracts). d Ensemble mean squared displacement (MSD) analysis derived from the single-particle tracking. Shaded area, standard error of the mean (SEM). Dashed lines are fitting to MSD(t)=Γtα, where Γ is the generalized diffusion coefficient34. The fitted {Γ, α} for disorganized and organized cytoplasm are {1.12, 0.75}, and {0.65, 0.78}, respectively. The localization error of these SPT experiments, quantified with immobilized microspheres, was below 0.01 µm2 (Supplementary Fig. 1a). e, f Representative FCS data of BSA-Alexa Flour 488 in cytoplasmic extracts. The full intensity trajectory (60 s) is shown in Supplementary Fig. 2c. The inset depicts a molecule diffusing through a confocal spot. G(τ) is the autocorrelation function, where τ is the lag time; δI(t)=I(t)−⟨I(t)⟩, where I(t) is the fluorescence intensity and the brackets denote time averages. The autocorrelation curve was fitted by an anomalous diffusion model (dashed line). In this example, the number of particles was 42, and the average intensity (shown in e) was 32 kHz, corresponding to a molecular brightness of 0.76 kHz/molecule.


Figure 2. a, Spatial regions of self-organization (color-coded), classified by the ER morphology. The dark sphere near the center of the inner interior is the nuclei. The image was stitched from multiple confocal images. b Representative confocal images of FCS foci, which are marked by the crosshairs. The annotated numbers refer to snapshots taken prior to FCS measurements in c. c Tracing protein diffusion during self-organization by FCS. The plots show the characteristic diffusion times τD and the diffusion mode α obtained from fitting the FCS curves. Black and gray dashed lines correspond to averages from disordered cytoplasmic extracts (in this example, 740 µs, Deff = 11.9 µm2/s) and cytosolic extracts (360 µs, Deff = 24.7 µm2/s), respectively. d Benchmark measurements from cytosolic extracts and buffers. ELB, egg lysis buffer; PBS, Phosphate-buffered saline. Insets show zoom-in of the data. Each condition was measured five times; all data presented by error bars are mean ± SEM. n = 5 measurements of different positions in a sample. e, Biological repeat of c, prepared from a separate batch of eggs. f, g are isolated traces from c and e. Error bars, estimated errors (standard deviation) from curve fitting (each fitting was performed over n = 107 data points). Scale bars, 100 µm.


Figure 6. a Schematic of the model: molecular and organelle reorganization optimizes molecular diffusion by creating longer mean free paths for diffusing molecules. The green object represents a protein of interest diffusing in the cytoplasm; the blue objects represent obstacles in the cytoplasm, such as organelles, cytoskeleton elements, and macromolecular complexes. In this model, proteins in organized cytoplasm remain free to diffuse in all directions. b Diffusion effects of molecular reorganization illustrated with mean squared displacement, MSD (t) = Γtα. The parameters {Γ, α} estimated for disorganized cytoplasm, organized cytoplasm and the cytosol are {8, 0.75}, {8.25, 0.70}, and {30, 0.85}, respectively. Note that based on the tradeoff between diffusion times and subdiffusivity in the organizing cytoplasm (Fig. 3a), we have assumed that the acceleration of protein movements over these distance scales comes at a cost of slower dynamics over longer distance scales. We also limit the comparison around the length scale of FCS measurements (~0.1–0.5 µm) since protein dynamics may show distinct characteristics at shorter and longer distance scales.



Adapted with permission from Springer Nature on behalf of Nature Communications: Huang et al. (2022). Cytoplasmic organization promotes protein diffusion in Xenopus extracts. Nat Commun. 2022 Sep 23;13(1):5599. doi: 10.1038/s41467-022-33339-0.

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Last Updated: 2022-12-01