The diffusivity of macromolecules in the cytoplasm of eukaryotic cells varies over orders of magnitude and dictates the kinetics of cellular processes. However, a general description that associates the Brownian or anomalous nature of intracellular diffusion to the architectural and biochemical properties of the cytoplasm has not been achieved. Here we measure the mobility of individual fluorescent nanoparticles in living mammalian cells to obtain a comprehensive analysis of cytoplasmic diffusion. We identify a correlation between tracer size, its biochemical nature and its mobility. Inert particles with size equal or below 50 nm behave as Brownian particles diffusing in a medium of low viscosity with negligible effects of molecular crowding. Increasing the strength of non-specific interactions of the nanoparticles within the cytoplasm gradually reduces their mobility and leads to subdiffusive behaviour. These experimental observations and the transition from Brownian to subdiffusive motion can be captured in a minimal phenomenological model.
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We acknowledge M. Coppey-Moisan, B. Goud and S. Letard and P. Dubreuil for supplying various cells (see text). E.B. acknowledges the Ecole Doctorale Frontières du Vivant (FdV)—Programme Bettencourt for financial support. M.D. and M.C. acknowledge financial support from the French National Research Agency (ANR) Paris-Science-Lettres Program (ANR-10-IDEX-0001-02 PSL), Labex CelTisPhyBio (ANR-10-LBX-0038), the Human Frontier Science Program (grant no. RGP0005/2007) and the France-BioImaging infrastructure supported by ANR Grant ANR-10-INSB-04 (Investments for the Future). This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 686841 (MAGNEURON).
The authors declare no competing interests.
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Supplementary Video Legends 1–5, Supplementary Figures 1–17, Supplementary Tables 1–3
Life Sciences Reporting Summary
Representative video of 25 nm Rho-NPs diffusing in a HeLa cell after pinocytic loading. Three unburst vesicles can be seen at the top of the images: two very, close together, on the left and one, very bright, in the middle. These unburst vesicles are much larger than individual NPs and are basically immobile. Images were acquired every 10 ms, the video is encoded at 30 frames per second (fps). Scale bar represents 10 μm
Representative videos of 25 nm Rho-NPs diffusing in HeLa cells after pinocytic loading. Top row: crops of the field of view showing unburst vesicles. NPs encapsulated within these intact vesicles move very fast and cannot be tracked individually. Bottom row: examples of typical regions of interest chosen for NPs single-particle tracking analysis and selected in a way such that unburst vesicles were systematically excluded. The marked difference in the behaviour of free versus encapsulated NPs ensures a reliable discrimination between the two cases. Images were acquired every 10 ms, the video is encoded at 30 fps. Scale bars represent 1 μm
Representative video of QDs (QDs-PEG-NH2) diffusing in a HeLa cell after pinocytic loading. QDs clearly show on-off fluctuations (blinking) of the fluorescence signal (use Supplementary Fig. 4 as reference to more easily spot blinking particles). The blinking behaviour is a typical signature of individual emitters and indicates that QDs are mono-dispersed, confirming their escape from pinocytic vesicles and dismissing potential aggregations. Images were acquired every 30 ms, the video is encoded at 15 fps. The counter indicates the frame number. Scale bar represents 2 μm
Representative video of the simultaneous diffusion of two different types of QDs, QDs-605 emitting at 605 nm (green) and QDs-PEG-NH2 emitting at 655 nm (red), after concomitant pinocytic internalization. The images clearly illustrate that in the two HeLa cells visible in the video there is no co-localization of the two probes, on the contrary of what expect if NPs would have been still encapsulated inside unburst vesicles. Images were acquired every 30 ms, the video is encoded at 15 fps. Scale bar represents 5 μm
The video shows the concurrent detection of the two types of QDs displayed in Supplementary Video 4: QDs-605 (red circles) and QDs-PEG-NH2 (yellow circles). The images put well in evidence the absence of correlated dynamics in the mobility of the two probes, on the contrary of what expected if NPs would have been still encapsulated inside unburst vesicles. Images were acquired every 30 ms, the video is encoded at 10 fps. Scale bar represents 5 μm
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Etoc, F., Balloul, E., Vicario, C. et al. Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells. Nature Mater 17, 740–746 (2018). https://doi.org/10.1038/s41563-018-0120-7
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