Liquid–liquid phase separation is an important mechanism by which eukaryotic cells functionally organize their intracellular content and has been related to cell malignancy and neurodegenerative diseases. These cells also undergo ATP-driven mechanical fluctuations, yet the effect of these fluctuations on the liquid–liquid phase separation remains poorly understood. Here, we employ high-resolution microscopy and atomic force microscopy of live Jurkat T cells to characterize the spectrum of their mechanical fluctuations, and to relate these fluctuations to the extent of nucleoli liquid–liquid phase separation (LLPS). We find distinct fluctuation of the cytoskeleton and of the cell diameter around 110 Hz, which depend on ATP and on myosin activity. Importantly, these fluctuations negatively correlate to nucleoli LLPS. According to a model of cell viscoelasticity, we propose that these fluctuations generate mechanical work that increases intracellular homogeneity by inhibiting LLPS. Thus, active mechanical fluctuations serve as an intracellular regulatory mechanism that could affect multiple pathophysiological conditions.
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This research was supported by Grant no. 1761/17 from the Israeli Science Foundation. We thank Naomi Book (The Silberman Institute at HUJI) for her assistance with confocal microscopy.
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Supplementary material 2 (TIFF 311 kb). Proposed model for cell mechanical control of its LLPS state. Local and coordinated fluctuations in Ca++ (orange arcs) are suspected to occur adjacent to the cortical actin and to produce corresponding global contracture of the cortical acto-myosin mesh, due to actin–myosin interactions. These contractions result in synchronized longitudinal tension waves in the cortical actin (red arrows), and produce radial intracellular pressure waves (dotted red arrows) that decrease the LLPS state (green patches and dotted green arrows). These synchronized longitudinal tension waves of cortical actin also reduce the thermal-induced mobility of cortical actin.
Supplementary material 3 (TIFF 272 kb). Control x-t measurements using Tetraspeck microspheres. (a) Confocal image of 100nm Tetraspeck fluorescence microspheres Scale bar - 5 μm. (b) Confocal x-t image of two Tetraspeck microspheres. (c) The average power spectrum of the time-dependent changes in the distance between microsphere pairs (lower panel; N=12 pairs of microspheres). The results are compared with the power spectrum of the cell diameter, as shown in Fig. 5c (shown also in upper panel for convenience). All powers (including Fig 5c) were normalized according to powers of other frequencies except 110-145Hz. The normalized powers in the range of 110-145Hz of the control Tetraspeck microspheres were not significantly higher in comparison to other frequency, while the normalized powers in that range in normal non-treated cells were higher in compare to ATP-depleted cells (P=0.003) and other frequencies (P=0.04).
Supplementary material 4 (TIFF 261 kb). Windowed DFT of the fluctuations in cell stiffness as measured by AFM in live cells. Ten consecutive windows (for 10 depths of indentation) of the average power spectrum of the vibrations in stiffness. Data is shown for each frequency and for each group of cells as follows: (a) represents the spectra for live cells without ATP depletion (N=15) for the 10 windows of the depth of indentations and (b) represents the spectra after ATP depletion (N=15) for the 10 windows of the depth of indentations.
Supplementary material 5 (TIFF 131 kb). Temporal correlation analysis (OTICS) of the cell membrane. (a) Fluorescent microscopy imaging of a live Jurkat cell, stained with anti-CD45 antibody. Scale bar - 5 μm (cell is a representative of n=13). (b) The one-time-lag correlations shown in panel b (OTICS), for multiple cells (n=13) before and after ATP depletion.
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Wohl, I., Yakovian, O., Razvag, Y. et al. Fast and synchronized fluctuations of cortical actin negatively correlate with nucleoli liquid–liquid phase separation in T cells. Eur Biophys J 49, 409–423 (2020). https://doi.org/10.1007/s00249-020-01446-9
- Cortical actin
- Liqud–liquid phase separation
- DFT analysis
- Plasma membrane