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.
Optical trapping and laser interferometry enable the non-invasive manipulation of colloids, which can be used to investigate the microscopic mechanics of surrounding media or bound macromolecules. For efficient trapping and precise tracking, the sample media must ideally be homogeneous and quiescent whereas such conditions are usually not satisfied in vivo in living cells. In order to investigate mechanics of the living-cell interior, we introduced (1) the in-situ calibration of optical trapping and laser interferometry, and (2) 3-D feedback control of a sample stage to stably track a colloidal particle. Investigating systematic errors that appear owing to sample heterogeneity and focal offsets of a trapping laser relative to the colloidal probe, we provide several important caveats for conducting precise optical micromanipulation in living cells. On the basis of this study, we further improved the performance of the techniques to be used in cells, by optimizing the position sensitivity of laser interferometry and the stability of the feedback simultaneously.
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