Cytoplasmic Anomalies: A heated debate about Phase Transitions as predictors of cellular states and disease

by Dr. Andres F Diaz

The second decade of the 21st century set the dawn of a well-announced rise in Cell Biophysics. Driven by innovation in microscopy methods and bridged with theoretical ideas from physics in vivo and in vitro. Cell Biophysics fertilized the ground for the idea that the cytoplasm is a highly dynamic environment, that could be experimentally de-constructed using in-vitro/in-vivo assays.

Cytoplasmic depictions in the late 20th century described the cell as composed of granular particles, punctae, or dots associated with a cellular structure linked to pathology. Often devoid of causal mechanisms, observations were supported by correlates in protein expression and phenomenological counts.

Although many of these depictions aided to discover pathways involved in certain pathologies, they were silently adopted as a general model on how cells worked fundamentally, driven by sequence steps in model pathway graphs. Such molecular models of a cell, would unequivocally lead to seek for targets limiting homeostatic conditions in cells hampering the signal transduction process of (Adenosine Triphosphate) ATP or (Guanosine Triphosphate) GTP leading to pathologies in the form of aggregates as a malfunction.

Cells surely utilize chemical energy but have more sophisticated ways to exploit it rather than specifically targeting pathways in a graph. For instance, rapid responses to most environmental conditions do not occur following a stepwise dynamics but instead, diffusive fluxes within cells can trigger the activation or suppression of a plethora of such pathways simultaneously. This is possible by leveraging the influence of molecular noise and diffusion, that can efficiently actuate upon molecular "gears" mechanisms that build rapid and robust responses within cells that otherwise would take a huge amount of energy to mount. For example, transcription, RNA silencing, and chromatin folding, thermal stress among others.

The question of whether cells can actively regulate and control every step reaction during their life-time and make use of molecular noise to leverage simultaneous responses, has particular value when predicting the outcome of cytosolic states, for example, senescence, quiescence, dormancy, death, and disease.

The debate heats up among scientists from all disciplines when asked, Can we understand cells as thermodynamical entities? and what enables cells to maintain parallel control of their transduction pathways?. The idea that cells use available thermal energy to drive their constituent pathways, had been disregarded by Biologists for years, mainly due to the belief that cells are mysterious entities that escape the laws of thermodynamics and that evolution has shaped such pathways in exquisite specificity, therefore not to be confused with a simple steam engine.

Despite this blind disbelief, experimental evidence suggests that temperature globally acts in a predictable and reversible manner upon the functioning of cell cycle progression, mainly by accelerating or decelerating the rates at which it occurs.

A classic and more consistent example of this is P granules. First described in Caenorhabditis elegans, P granules are part of a wide family of molecular condensates responsible for RNA processing that in the nematode, participate in RNA sequestration and transport to the germline or the cells responsible to produce gametes. 

P granules were described as puncta via immunofluorescence, their appearance like fluorescent grains remained unquestioned for several decades although, the mechanisms of how such compartments formed and were transported remained unknown.

A major breakthrough came with the discovery of protein condensates in the nematode worm C. elegans in 2009 by Cliff Brangwynne at Anthony Hyman's lab in Dresden - Germany. Brangwyne showed that such granules were not solid but resembled viscosity and were poised to dissolve and condense within minutes, and were susceptible to deformation showing to associated surface tension in the absence of a membrane-like compartment. 

P granules ghostly nature showed breathtaking properties, that challenged the static step-wise depictions of how proteins could work in a cellular liquid environment. The strongest most important demonstration of this behavior was that P granule's chemical potential showed to be continuous in space-time. Or, that molecules within would remain unchanged despite their size or position. In other words "some inner potential" was able to drive molecules to continuously concentrate and turn-over at nearly constant rates within the condensate.

The recovery of fluorescence occured in milisenconds in ATP/GTP deprived media, demonstrating in-vivo and in-vitro that chemical energy was not required for their co-existance with the liquid surrounding. This settled down the ground for a continuous stream of studies on LPS composition and dynamics of P granules in-vivo and in-vitro, but also of other proteins such as FUS involved in Amyotrophic Lateral Sclerosis (ALS) granting them the name of phase-separated protein condensates.

Brangwyne studies went beyond C. elegans to understand whether other structures shared such behavior in X. laevis, with much larger cell sizes, and found that nucleoli, also shared such properties and furthermore scaled their size with membrane-bound compartments such as the nucleus.

Such experiments presented new evidence, that protein concentration and diffusion, are major drivers of macroscopic manifestation of intermolecular molecular forces that do not necessarily utilize chemical energy to form. These molecular condensates showed evidence that Liquid Phase Separation (LPS) can occur in living cells and that this process requires physical favorable interactions to occur.

It is clear today that protein diffusion, molecular volume, and weak intermolecular interactions participate in (LPS). Scientists today all over the world have learned via in-vitro and in-vivo experiments, that ionic strength of other solutes such as salts, RNA and DNA can regulate LPS, contributing to the idea that the cytosol and its components can be de-constructed and studied, at least for now, in a qualitative manner as if it were a thermodynamical system or a steam engine that could be dismantled and re-built.

The importance of considering the cytosol as a thermodynamical system is that it is possible to predict whether the cytosol is "hot/cold" or favorably "close/far" from a given "state" poised for condensation or dissolution. 

The relevance of these investigations, enable us to develop models that better allow us to predict when and how a cell may undergo a cytoplasmic transformation such as a phase transition, and whether or not this phase transition results in pathology or not.

To achieve such a level of sophistication in predicting global cytoplasmic states in a qualitative manner, careful experimental measurements started to describe in 2013 until today, in Max Planck CBG in Dresden and Max Planck PKS by describing for the first time a Thermally Driven Phase Transition in a living cell. 

In the future, with more advanced techniques in microscopy, nanotechnolgy and biochemistry, it is intended to comprehend the diversity of states that the cytoplasm of cells are able to execute thermodynamically speaking, opening the gates to a better understanding of how multicomponent thermal systems work.