Thermorheology of living cells: impact of temperature variations on cell mechanics

Upon temperature changes, we observe a systematic shift of creep compliance curves J (t) for single living breast epithelial cells. We use a dual-beam laser trap (optical stretcher) to induce temperature jumps within milliseconds, while simultaneously measuring the mechanical response of whole cells to optical force. The cellular mechanical response was found to differ between sudden temperature changes compared to slow, long-term changes implying adaptation of cytoskeletal structure. Interpreting optically induced cell deformation as a thermorheological experiment allows us to consistently explain data on the basis of time–temperature superposition, well known from classical polymer physics. Measured time shift factors give access to the activation energy of the viscous flow of MCF-10A breast cells, which was determined to be 80 kJ mol−1. The presented measurements highlight the fundamental role that temperature plays for the deformability of cellular matter. We propose thermorheology as a powerful concept to assess the inherent material properties of living cells and to investigate cell regulatory responses upon environmental changes

Predicting disordered regions driving phase separation of proteins under variable salt concentration

We determine the intrinsically disordered regions (IDRs) of phase separating proteins and investigate their impact on liquid-liquid phase separation (LLPS) with a random-phase approx- imation (RPA) that accounts for variable salt concentration. We focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that an IDR near the C-terminus pro- motes LLPS, which we validate through direct comparison with in vitro experimental results. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS is not as well understood. Apart from the LC domain we here identify two IDRs, one near the N-terminus and another near the C-terminus. Our RPA analysis of these domains predict that, surprisingly, the IDR at the N-terminus (aa 1-285) and not the LC domain promotes LLPS of FUS by comparison to in vitro experiments under physiological temperature and salt conditions

Thermal instability of cell nuclei

DNA is known to be a mechanically and thermally stable structure. In its double stranded form it is densely packed within the cell nucleus and is thermo-resistant up to 70 °C. In contrast, we found a sudden loss of cell nuclei integrity at relatively moderate temperatures ranging from 45 to 55 °C. In our study, suspended cells held in an optical double beam trap were heated under controlled conditions while monitoring the nuclear shape. At specific critical temperatures, an irreversible sudden shape transition of the nuclei was observed. These temperature induced transitions differ in abundance and intensity for various normal and cancerous epithelial breast cells, which clearly characterizes different cell types. Our results show that temperatures slightly higher than physiological conditions are able to induce instabilities of nuclear structures, eventually leading to cell death. This is a surprising finding since recent thermorheological cell studies have shown that cells have a lower viscosity and are thus more deformable upon temperature increase. Since the nucleus is tightly coupled to the outer cell shape via the cytoskeleton, the force propagation of nuclear reshaping to the cell membrane was investigated in combination with the application of cytoskeletal drugs

Complex thermorheology of living cells

Temperature has a reliable and nearly instantaneous influence onmechanical responses of cells.As recently published, MCF-10Anormal epithelial breast cells follow the time–temperature superposition (TTS) principle. Here,wemeasured thermorheological behaviour of eightcommoncell types within physiologically relevant temperatures and appliedTTS to creep compliance curves.Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C.Activation energies (EA)were calculated for all cell types and ranged between 50 and 150 kJmol−1.The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes aswell as structural composition of the cells in response tomechanical load and temperature increase.This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlledwhen designing experiments

Testing the differential adhesion hypothesis across the epithelial− mesenchymal transition

Weanalyze the mechanical properties of three epithelial/mesenchymal cell lines (MCF-10A, MDAMB- 231, MDA-MB-436) that exhibit a shift in E-, N- and P-cadherin levels characteristic of an epithelial−mesenchymal transition associated with processes such as metastasis, to quantify the role of cell cohesion in cell sorting and compartmentalization. Wedevelop a unique set of methods to measure cell–cell adhesiveness, cell stiffness and cell shapes, and compare the results to predictions from cell sorting in mixtures of cell populations.Wefind that the final sorted state is extremely robust among all three cell lines independent of epithelial or mesenchymal state, suggesting that cell sorting may play an important role in organization and boundary formation in tumours.Wefind that surface densities of adhesive molecules do not correlate with measured cell–cell adhesion, but do correlate with cell shapes, cell stiffness and the rate at which cells sort, in accordance with an extended version of the differential adhesion hypothesis (DAH). Surprisingly, theDAHdoes not correctly predict the final sorted state. This suggests that these tissues are not behaving as immiscible fluids, and that dynamical effects such as directional motility, friction and jamming may play an important role in tissue compartmentalization across the epithelial−mesenchymal transition

Cell membrane softening in human breast and cervical cancer cells

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells

Thermorheology of living cells: impact of temperature variations on cell mechanics

Upon temperature changes, we observe a systematic shift of creep compliance curves J (t) for single living breast epithelial cells. We use a dual-beam laser trap (optical stretcher) to induce temperature jumps within milliseconds, while simultaneously measuring the mechanical response of whole cells to optical force. The cellular mechanical response was found to differ between sudden temperature changes compared to slow, long-term changes implying adaptation of cytoskeletal structure. Interpreting optically induced cell deformation as a thermorheological experiment allows us to consistently explain data on the basis of time–temperature superposition, well known from classical polymer physics. Measured time shift factors give access to the activation energy of the viscous flow of MCF-10A breast cells, which was determined to be 80 kJ mol−1. The presented measurements highlight the fundamental role that temperature plays for the deformability of cellular matter. We propose thermorheology as a powerful concept to assess the inherent material properties of living cells and to investigate cell regulatory responses upon environmental changes

Thermorheology of living cells: impact of temperature variations on cell mechanics

Upon temperature changes, we observe a systematic shift of creep compliance curves J (t) for single living breast epithelial cells. We use a dual-beam laser trap (optical stretcher) to induce temperature jumps within milliseconds, while simultaneously measuring the mechanical response of whole cells to optical force. The cellular mechanical response was found to differ between sudden temperature changes compared to slow, long-term changes implying adaptation of cytoskeletal structure. Interpreting optically induced cell deformation as a thermorheological experiment allows us to consistently explain data on the basis of time–temperature superposition, well known from classical polymer physics. Measured time shift factors give access to the activation energy of the viscous flow of MCF-10A breast cells, which was determined to be 80 kJ mol−1. The presented measurements highlight the fundamental role that temperature plays for the deformability of cellular matter. We propose thermorheology as a powerful concept to assess the inherent material properties of living cells and to investigate cell regulatory responses upon environmental changes

Thermal instability of cell nuclei

DNA is known to be a mechanically and thermally stable structure. In its double stranded form it is densely packed within the cell nucleus and is thermo-resistant up to 70 °C. In contrast, we found a sudden loss of cell nuclei integrity at relatively moderate temperatures ranging from 45 to 55 °C. In our study, suspended cells held in an optical double beam trap were heated under controlled conditions while monitoring the nuclear shape. At specific critical temperatures, an irreversible sudden shape transition of the nuclei was observed. These temperature induced transitions differ in abundance and intensity for various normal and cancerous epithelial breast cells, which clearly characterizes different cell types. Our results show that temperatures slightly higher than physiological conditions are able to induce instabilities of nuclear structures, eventually leading to cell death. This is a surprising finding since recent thermorheological cell studies have shown that cells have a lower viscosity and are thus more deformable upon temperature increase. Since the nucleus is tightly coupled to the outer cell shape via the cytoskeleton, the force propagation of nuclear reshaping to the cell membrane was investigated in combination with the application of cytoskeletal drugs

Complex thermorheology of living cells

Temperature has a reliable and nearly instantaneous influence onmechanical responses of cells.As recently published, MCF-10Anormal epithelial breast cells follow the time–temperature superposition (TTS) principle. Here,wemeasured thermorheological behaviour of eightcommoncell types within physiologically relevant temperatures and appliedTTS to creep compliance curves.Our results showed that superposition is not universal and was seen in four of the eight investigated cell types. For the other cell types, transitions of thermorheological responses were observed at 36 °C.Activation energies (EA)were calculated for all cell types and ranged between 50 and 150 kJmol−1.The scaling factors of the superposition of creep curves were used to group the cell lines into three categories. They were dependent on relaxation processes aswell as structural composition of the cells in response tomechanical load and temperature increase.This study supports the view that temperature is a vital parameter for comparing cell rheological data and should be precisely controlledwhen designing experiments