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Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity

Abstract

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus is spatially heterogeneous at the nanoscale, and that variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the organization of the genome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the rate is determined by the specific subcellular environments. We thus unveil rich heterogeneities and charge effects in intracellular diffusion at the nanoscale.

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Fig. 1: SMdM for single mEos3.2 FP molecules freely diffusing in the cytoplasm of live mammalian cells.
Fig. 2: SMdM of free mEos3.2 in the mammalian cytoplasm and correlated SMLM of the actin cytoskeleton.
Fig. 3: SMdM of free mEos3.2 in the nucleus, and correlated SMLM of DNA.
Fig. 4: SMdM of mEos3.2 species of different net charges.
Fig. 5: SMdM of different +7-charged species.
Fig. 6: SMdM of the nonphotoswitchable GFP mEmerald in the mammalian cytoplasm.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The custom codes for the data analysis used in this study are available from the corresponding author upon request.

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Acknowledgements

We thank S. Moon for discussion, and M. He and Y. Shyu for help with preparation of the DNA constructs. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (grant no. DP2GM132681), the Beckman Young Investigator Program and the Packard Fellowships for Science and Engineering to K.X. K.X. is a Chan Zuckerberg Biohub investigator.

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K.X. conceived the research. L.X. and K.C. designed and conducted the experiments. All authors contributed to experimental designs, data analysis and paper writing.

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Correspondence to Ke Xu.

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Extended data

Extended Data Fig. 1 SMdM results at different single-molecule densities.

Free mEos3.2 was expressed in the cytoplasm of a PtK2 cell, and SMdM was performed on the same cell at a low single-molecule density of ~0.05 molecules/µm2/frame for 60,000 pairs of pulses (ac), or at a high single-molecule density of ~0.11 molecules/µm2/frame for 30,000 pairs of pulses (df) by increasing the power of the photoactivation (405 nm) laser. (a) SMdM diffusivity map for the low single-molecule density experiment, obtained by spatially binning the single-molecule displacement d data onto 120×120 nm2 grids, and then individually fitting the distribution of d in each bin to Eq. 2 through MLE. (b, c) Distribution of 1-ms single-molecule displacement for two 360×360 nm2 areas inside (b; white arrow in a) and outside (c; red arrow in a) a linear structure of reduced local diffusivity, respectively. Blue lines are MLE results using Eq. 2, with resultant D and uncertainty σ labeled in each panel. (df) Results of the high single-molecule density experiment: comparable D values are obtained with the much-reduced number of pulse pairs, despite an increased background due to single-molecule mismatch. These experiments were independently repeated 10 times with similar results.

Extended Data Fig. 2 Additional SMdM results of free mEos3.2 in the cytoplasm of live U2OS and PtK2 cells, and correlated SMLM of the actin cytoskeleton.

(a, b) Correlated SMdM diffusivity map for a live U2OS cell (a) vs. SMLM image of Alexa Fluor 647 phalloidin-labeled actin in the fixed cell (b). (a) and (b) were independently repeated 4 times with similar results. (c, d) Additional SMdM diffusivity maps for the cytoplasm of PtK2 cells. (c) and (d) were independently repeated 11 times with similar results.

Extended Data Fig. 3 Additional SMdM results of free mEos3.2 in the nuclei of live PtK2 cells, and correlated SMLM of DNA.

(a, d, f) SMdM diffusivity maps of 3 different cells. (b) Bright-field transmission image of the same view as (a), visualizing the nucleolus. (c, e, g) SMLM images of the fixed cells in (a, d, f) using the DNA stain NucSpot Live 650. We note that as the SMLM of DNA was performed after fixation and multiple washing steps, it was difficult to image at exactly the same focal plane as the live-cell SMdM experiment, which accounts for some of the apparent structural mismatches. Scale bars in all panels: 2 µm. These experiments were independently repeated 23 times with similar results.

Extended Data Fig. 4 SMdM of mEos3.2-NLS and correlated SMLM of DNA.

(a) SMdM diffusivity map of mEos3.2-NLS in the nucleus of a live PtK2 cell. (b) SMLM image of the fixed cell using the DNA stain NucSpot Live 650. (c) Overlay of (a) and (b). Scale bars: 2 µm. These experiments were independently repeated 18 times with similar results.

Extended Data Fig. 5 SMdM of free mEos3.2 using 488 nm excitation without photoactivation.

(a, b) SMdM diffusivity maps of mEos3.2-C1 in the cytoplasm of live PtK2 cells, obtained by exciting the un-photoconverted, “green” form of mEos3.2 single molecules with 488 nm excitation. (a) and (b) were independently repeated 6 times with similar results. (c, d) Distribution of 1-ms single-molecule displacement for two 360×360 nm2 areas inside (c; white arrow in a) and outside (d; red arrow in a) a linear structure of reduced local diffusivity, respectively. Blue lines are MLE results using Eq. 2, with resultant D and uncertainty σ labeled in each panel.

Extended Data Fig. 6 Asymmetric effects of negative and positive net charges on intracellular diffusion.

(a) A negatively charged diffuser is readily neutralized by the abundant, small metal cations inside the cell, and so diffuses similarly as neutral counterparts. (b) A positively charged diffuser is not effectively neutralized/screened by the very limited amount of intracellular small anions; its dynamic interactions with the negatively charged, large biomolecules insides the cell substantially hinder diffusion.

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Xiang, L., Chen, K., Yan, R. et al. Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity. Nat Methods 17, 524–530 (2020). https://doi.org/10.1038/s41592-020-0793-0

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