Thermodynamic equilibrium is an idealized, theoretical state that can be closely approximated in certain controlled environments. Although conceptually interesting, the vast majority of large-scale or dynamic systems in the universe are considered non-equilibrium, meaning they are characterised by a continuous flow of energy and matter. Living systems are no exception, as they constantly consume and dissipate energy. For scientists to explore the dynamic nature of living cells, methods to study deviations from equilibrium are essential. In a new study published in PRX, scientists from the Fischer-Friedrich group at the Cluster of Excellence Physics of Life (PoL) sought to study these hallmarks of life. Their findings challenge a widely accepted notion amongst biophysicists.
The authors used HeLa cells, a widely studied human cancer cell line, as a simplified physical model. Focusing on the actin cortex—a dynamic, thin, mesh-like network of structural proteins just beneath the cell membrane—they measured the cell’s internal noise fluctuations. After arresting cells in mitosis, they probed fluctuations in the cortex using atomic force microscopy (AFM), a highly sensitive technique for measuring tiny cellular forces. “To give an analogy, our approach is similar to sensing vibrations with a fingertip. The AFM tip can detect fluctuations as small as a few nanometres in amplitude,” explained Dr. Narinder, the lead author of the study.
The authors observed that these fluctuations were significantly amplified compared to the surrounding environment. This amplification, often measured by physicists as effective temperature (Teff), has been used to quantify distance from thermal equilibrium for decades. Inside a cell, active components like molecular motors are adding dynamics to the cortex which cause fluctuations or ‘noise’ greater than simple thermal motion. The prevailing hypothesis in the field has been that any measures quantifying non-equilibrium dynamics using effective temperature, and entropy production should follow common trends. However, the actin cortex presents a more intricate scenario, as dynamics are influenced not only by motor proteins, but also by continuous turnover of actin filaments. These processes, which happen simultaneously, can introduce complex fluctuations that make standard measurements difficult to interpret.
To isolate and understand the effects of both processes in the cortex, the authors carried out a targeted approach: they introduced two key perturbations into the system: inhibiting molecular motors (like myosin) and inhibiting the cell’s ability to process actin polymer turnover. After treating the cells with inhibitors, the authors measured fluctuations using AFM and compared the readout to controls. Inhibiting motor proteins resulted in significant decreases in effective temperature, highlighting how these proteins drive active fluctuations in the cell. Crucially, the experiment revealed that when protein turnover is inhibited (making the cell more solid-like or elastic), the resulting change in physical properties kept Teff high, but it drastically reduced the energy dissipation and, consequently, entropy production. This finding highlighted that Teff may not be as reliable as a metric as previously thought: the amplified fluctuations it reflects can be tuned independently of the processes that drive cells out of equilibrium.
If Teff is not a useful signature for studying living systems, what could be used instead? According to Narinder: “We showed experimentally that these fluctuations are not necessarily related, rather it is the time irreversibility which reflects the degree of activity in the system”. Living cells are powered through constant energy consumption (with ATP), which means the internal dynamics are fundamentally time-asymmetric, meaning the probability of a movement occurring is not the same as the probability of the movement happening in reverse. An open question remains whether a law can be derived for living systems, that connects entropy production and time irreversibility in biological dynamics.
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Original publication
Narinder Narinder, Elisabeth Fischer-Friedrich. Time irreversibility, entropy production and effective temperature are independently regulated in the actin cortex of living cells. PRX (2025). DOI: https://doi.org/10.1103/5zyn-kgs3