Nishizawa K et al. (2017), Universal glass-forming behavior of in vitro an...

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Sci Rep 2017 Nov 09;71:15143. doi: 10.1038/s41598-017-14883-y.

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Universal glass-forming behavior of in vitro and living cytoplasm.

Nishizawa K , Fujiwara K , Ikenaga M , Nakajo N , Yanagisawa M , Mizuno D .

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Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of in vitro and living cytoplasm using the particle-tracking and manipulation technique. The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart.

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	Figure 1. Concentration dependence of viscosity. η/η w (η w: viscosity of water) for BSA solutions (red circles) and cell extracts (green triangles: E. coli, blue squares: Xenopus eggs, black diamonds: HeLa cells). The solid and broken lines are fits of Equation (1) to the BSA and cell extract data, respectively. Error bar in x-direction stem from estimation error of the concentration and Error bar in y-direction stem from the estimation error of the effect of laser trap. This effect of laser trap on viscosity become negligible when η/η w become large.
	Figure 2. Bead-pulling MR in force clamp mode. (a) A schematic of the particle-pulling experiment. A constant force was applied to the probe by maintaining a constant distance x between the probe center and laser focus. Resulting velocity v yields viscosity η. (b) The relation between the time and displacement in cell extracts prepared from E. coli at various concentrations [0.10 g/mL pulled by 0.25 pN (green), 0.14 g/mL by 0.19 pN (blue), and 0.21 g/mL by 0.14 pN (black)] and in BSA solutions [BSA 0.42 g/mL pulled by 0.18 pN (orange), and BSA 0.53 g/mL pulled by 0.18 pN (red)]. (c) The dependence of the applied pulling force on relative viscosity η/η w in BSA solutions (orange circles: 0.42 g/mL, and red filled circles: 0.53 g/mL) and in E. coli cell extracts (green triangles: 0.10 g/mL, blue filled triangles: 0.14 g/mL, and black filled triangles: 0.21 g/mL). (d) η pull of BSA solutions and IVCEs (E. coli) evaluated by particle pulling are plotted together with η estimated from PMR (solid curves).
	Figure 3. Angell plots. Relative viscosity η/η w as a function of scaled concentration c/c g for BSA (red circles and the dash-dot-dot curve), for cell extracts from E. coli (green triangles and the dotted curve), and for cytoplasm in a living cell (pink diamonds and the solid line). Curves are the fits of Equation (1); c g for each sample was determined as a concentration where η/η w becomes 10 5-fold greater than that in water. The solid curve represents the results reported in refs31,39 for the suspension of hard-sphere colloids of uniform size. The solid line indicates Arrhenius behavior for strong glass to which viscosity in living cells conforms.
	Figure 4. Diffusion in living cytoplasm and in vitro cytoplasm measured by FRAP. (a) Fluorescent images of GFP-labeled living spheroplasts of E. coli before, immediately after (t = 0 s), and 10 s after photobleaching. The photobleached region in a metabolically inactive spheroplast remained dark even at 10 s after photobleaching (lower panels), whereas the dark region was obscured already at t = 0 in normal spheroplasts (upper panels) because of rapid transport. (b) Inverse of diffusion coefficients D of GFP in IVCEs/liposomes (open triangles), IVCEs/emulsions (filled triangles) and BSA/liposomes (circles) measured with FRAP. These data were normalized to the value in water as D w/D. The broken and solid curves indicate η/η w in IVCEs and BSA solutions which are measured by PMR (same lines with that in Fig. 1), respectively. D w/D of GFP in normal (white bar) and metabolically inactive spheroplast cells (black bar) are also shown.
	Figure 5. Concentration dependence of viscosities at 4 kHz in living cells and in cell extracts. η @4kHz/η w for cell extracts (green triangles: E. coli, blue squares: Xenopus eggs, black diamonds: HeLa cells) and for cells (pink diamonds: HeLa cells, filled orange downward triangles: MDCK cells, purple crosses: NIH3T3 cells). The green solid line represents fit of Equation (1) to η ext@4kHz/η w measured by PMR. A solid pink line is the exponential fit for cell extracts at low concentration and living cells.
	Figure 6. The microrheology setup and trapping-force calibration. (a) A simplified schematic of the experimental setup. A constant trapping force was applied by rapidly steering the position of the drive laser (λ = 1064 nm) using the feedback-controlled AOD (force clamp). Displacements of the probe were detected using a fixed probe laser (λ = 830 nm). In the bead-pulling experiments, the piezo stage was also feedback controlled in order to eliminate the probe drift. (b) Imaginary parts of the response functions, A k (red curve), A 3k (blue curve), and α (black curve), measured in BSA solution (0.54 g/mL). Full complex responses including real parts are shown in Supplementary Fig. S10. With the increase of trap stiffness, the responses were suppressed at low frequencies.

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