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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Ambadipudi S, Biernat J, Riedel D et al (2017) Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nat Commun 8:1–13. https://doi.org/10.1038/s41467-017-00480-0
Bouchard JJ, Otero JH, Scott DC et al (2018) Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments. Mol Cell 72:19–36.e8. https://doi.org/10.1016/j.molcel.2018.08.027
Brangwynne CP, Mitchison TJ, Hyman AA (2011) Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc Natl Acad Sci 108:4334–4339. https://doi.org/10.1073/pnas.1017150108
Brangwynne CP, Tompa P, Pappu RV (2015) Polymer physics of intracellular phase transitions. Nat Phys 11:899–904. https://doi.org/10.1038/nphys3532
Buchanan PJ, McCloskey KD (2016) CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics. Eur Biophys J 45:621–633. https://doi.org/10.1007/s00249-016-1144-z
Chugh P, Paluch EK (2018) The actin cortex at a glance. J Cell Sci 131:jcs186254. https://doi.org/10.1242/jcs.186254
Derenzini M, Trerè D, Pession A et al (2000) Nucleolar size indicates the rapidity of cell proliferation in cancer tissues. J Pathol 191:181–186. https://doi.org/10.1002/(SICI)1096-9896(200006)191:2%3c181:AID-PATH607%3e3.0.CO;2-V
Donovan JA, Koretzky GA (1993) CD45 and the immune response. J Am Soc Nephrol 4:976–985
Gittes F, Schnurr B, Olmsted PD et al (1997) Microscopic viscoelasticity: shear moduli of soft materials determined from thermal fluctuations. Phys Rev Lett. https://doi.org/10.1103/PhysRevLett.79.3286
Guo M, Ehrlicher AJ, Jensen MH et al (2014) Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy. Cell 158:822–832. https://doi.org/10.1016/j.cell.2014.06.051
Hudder A, Nathanson L, Deutscher MP (2003) Organization of mammalian cytoplasm. Mol Cell Biol 23:9318–9326. https://doi.org/10.1128/MCB.23.24.9318-9326.2003
Hyman AA, Weber CA, Jülicher F (2014) Liquid-liquid phase separation in biology. Annu Rev Cell Dev Biol. https://doi.org/10.1146/annurev-cellbio-100913-013325
Ishijima A, Kojima H, Funatsu T et al (1998) Simultaneous observation of individual ATPase and mechanical events by a single myosin molecule during interaction with actin. Cell 92:161–171. https://doi.org/10.1016/S0092-8674(00)80911-3
Kang J, Lim L, Song J (2018) ATP enhances at low concentrations but dissolves at high concentrations liquid-liquid phase separation (LLPS) of ALS/FTD-causing FUS. Biochem Biophys Res Commun 504:545–551. https://doi.org/10.1016/j.bbrc.2018.09.014
Kindzelskii AL, Petty HR (2003) Intracellular calcium waves accompany neutrophil polarization, formylmethionylleucylphenylalanine stimulation, and phagocytosis: a high speed microscopy study. J Immunol. https://doi.org/10.4049/jimmunol.170.1.64
Kolin DL, Wiseman PW (2007) Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells. Cell Biochem Biophys 49:141–164. https://doi.org/10.1007/s12013-007-9000-5
Kostylev MA, Tuttle MD, Lee S et al (2018) Liquid and hydrogel phases of PrPC linked to conformation shifts and triggered by Alzheimer’s amyloid-β oligomers. Mol Cell 72:426–443.e12. https://doi.org/10.1016/j.molcel.2018.10.009
Louvet E, Junéra HR, Berthuy I, Hernandez-Verdun D (2006) Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process. Mol Biol Cell 17:2537–2546. https://doi.org/10.1091/mbc.E05-10-0923
Lukinavičius G, Reymond L, D’Este E et al (2014) Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11:731–733. https://doi.org/10.1038/nmeth.2972
Mackenzie IR, Nicholson AM, Sarkar M et al (2017) TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics. Neuron 95:808–816.e9. https://doi.org/10.1016/j.neuron.2017.07.025
Ming Y, Chen X, Xu Y et al (2019) Targeting liquid-liquid phase separation in pancreatic cancer. Transl Cancer Res 8:96–103. https://doi.org/10.21037/tcr.2019.01.06
Mizuno D, Tardin C, Schmidt CF, MacKintosh FC (2007) Nonequilibrium mechanics of active cytoskeletal networks. Science (80-) 315:370–373
Molliex A, Temirov J, Lee J et al (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163:123–133. https://doi.org/10.1016/j.cell.2015.09.015
Parry BR, Surovtsev IV, Cabeen MT et al (2014) The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity. Cell 156:183–194. https://doi.org/10.1016/j.cell.2013.11.028
Patel A, Lee HO, Jawerth L et al (2015) A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162:1066–1077. https://doi.org/10.1016/j.cell.2015.07.047
Perez-Reyes E (2015) Molecular physiology of low-voltage-activated T-type calcium channels. Physiol Rev 83:117–161. https://doi.org/10.1152/physrev.00018.2002
Protasi F (2019) Ryanodine receptors of striated muscles : a complex capable of multiple interactions. Physiol Rev 77:699–729
Saks V, Beraud N, Wallimann T (2008) Metabolic compartmentation—a system level property of muscle cells: real problems of diffusion in living cells. Int J Mol Sci 9:751–767. https://doi.org/10.3390/ijms9050751
Salbreux G, Charras G, Paluch E (2012) Actin cortex mechanics and cellular morphogenesis. Trends Cell Biol 22:536–545. https://doi.org/10.1016/j.tcb.2012.07.001
Shav-Tal Y, Blechman J, Darzacq X et al (2005) Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell 16:2395–2413. https://doi.org/10.1091/mbc.E04-11-0992
Snaar S, Wiesmeijer K, Jochemsen AG et al (2000) Mutational analysis of fibrillarin and its mobility in living human cells. J Cell Biol 151:653–662. https://doi.org/10.1083/jcb.151.3.653
Style RW, Sai T, Fanelli N et al (2018) Liquid-liquid phase separation in an elastic network. Phys Rev X 8:11028. https://doi.org/10.1103/PhysRevX.8.011028
Tuvia S, Levin S, Bitler A, Korenstein R (1998) Mechanical fluctuations of the membrane-skeleton are dependent on F- actin ATPase in human erythrocytes. J Cell Biol 141:1551–1561. https://doi.org/10.1083/jcb.141.7.1551
Vahabikashi A, Park CY, Perkumas K et al (2019) Probe sensitivity to cortical versus intracellular cytoskeletal network stiffness. Biophys J 116:518–529. https://doi.org/10.1016/j.bpj.2018.12.021
Wakui M, Potter BVL, Petersen OH (1989) Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration. Nature. https://doi.org/10.1038/339317a0
Wang YL, Lanni F, McNeil PL et al (1982) Mobility of cytoplasmic and membrane-associated actin in living cells. Proc Natl Acad Sci U S A 79:4660–4664. https://doi.org/10.1073/pnas.79.15.4660
Wohl I, Sherman E (2019) ATP-dependent diffusion entropy and homogeneity in living cells. Entropy 21:962–980. https://doi.org/10.3390/e21100962
Wohl I, Zurgil N, Hakuk Y et al (2017) Fluctuation of information entropy measures in cell image. Entropy. https://doi.org/10.3390/e19100565
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