Self-Organizing Molecules In Cells

The fantastic phenomenon of liquid-liquid phase separation.

Edited by: Nikolina Djuric

Photo by Landon Arnold on Unsplash

It is always fascinating to see how one foundation of a person’s understanding, whether in science or any other field, can so drastically experience a paradigm shift with a new perspective. I personally began this paradigm shift through an article in the Journal of Neuroscience (Vol. 41, Issue 5) published on February 3rd, 2021.

This paper blew my mind.

In this article, Hayashi et al. (2021) discuss liquid-liquid phase separation (LLPS), a phenomenon which can unravel many mysteries about cellular functions. LLPS is similar to an oil droplet in water, where the properties of liquids such as immiscibility and density keep the two from mixing. Oil is composed of particles that are nonpolar while water molecules are polar. This creates electrostatic conditions that favor the attractiveness between the oil molecules to each other, and water molecules to themselves — the elementary school adage of “like dissolves like”. These properties also influence the motion of particles based on their concentration.

In fact, this is the earliest record exploring the diffusion of water through a membrane that I could find on pubmed.

Jacques Loeb’s (1920) article describing the impact ionic charge and valency has on water diffusion through a membrane (published in The Journal of General Physiology, 20;2(4):387–408).

Loeb explains that, in an experimental condition where pure water is separated by a membrane from another solution, water diffuses into the solution while the solute diffuses out. The rate at which the solute moves into the pure water is proportional to the concentration of the solution (Loeb, 1920). Meanwhile, the diffusion of water into the solution has a different character depending on the electrochemical properties of the solute. A solute that is a non-electrolyte (electrically neutral) has water diffuse into it at a rate that is proportional to its concentration. However, when the solute carries some kind of charge, the water’s rate of diffusion shows anomalies in its behavior when the concentrations of the solute are particularly low.

Graphs generated by Jacques Loeb’s experiments (1920) showing how the initial rate of diffusion through a membrane increases with increasing valency of the anion of the electrolyte. The membrane through which the water diffused was also negatively charged, like the anion of the electrolyte.

The most fascinating aspect of Loeb’s studies is the hierarchy in the molecular phenomena that produce the non-linear curves in his graphs. There is the valency of ions, a property determined by the number of electrons on the outer shell of an atom or groups of atoms. There is the charge itself, positive for cations and negative for anions. And finally, there are the “pushing and pulling” effects of forces such as pressure.

The diversity of phenomena at play in this simple and clean experiment has breathed new life into the current model of cells.

Traditionally, we think of cellular function as depending on compartmentalization, which has probably been dictated by our ability to visualize membranous structures. The classical view creates an organized model of the cell; rather than being just a soup of things floating around, they are actually neatly packaged items that are shuttled to the regions of the cell where they are needed.

For example, in a neuron, neurotransmitters need to be packaged into vesicles (a membrane-bound compartment) and transported to the synapse of a presynaptic neuron. At the synapse, they can be released to diffuse across, and exert effects on, the postsynaptic neuron, propagating signals throughout the nervous system.

The fascinating thing about this example is the plethora of molecular events occurring that do not involve any particularly obvious structure. There are proteins that tether the vesicle to the membrane of the presynaptic neuron, which involves recognition of the vesicle and the connection between it and the “anchor”. There is the process of recognizing the signal for its release which triggers the process of bringing the vesicle towards the membrane of the presynaptic neuron. At this zone, it fuses with the cellular membrane and releases its contents into the synapse. Finally, there is the recognition of the contents on the postsynaptic neuron. None of these processes are random, chaotic events, but targeted and triggered activities created by the molecular entities in the vicinity of the vesicle. Ultimately, the effect is an increased probability of its release.

A neuronal synapse. Original Text: “This is a diagram of a typical central nervous system synapse. The presynaptic and postsynaptic neuron are on top and bottom. Mitochondria are light green, receptors dark green, postsynaptic density is in grey, Brown pyramids represent protein clusters composing the active zone, cell adhesion molecules are brown rectangles, synaptic vesicles are tan spheres, endoplasmic reticulum is the tan structure on the bottom left.” From Wikimedia Commons: Original drawing by Curtis Neveu.

But many questions arise along this line of thought. If molecules do not necessarily need to exist in a compartment, then they can also be “floating around”. How would a high concentration of molecules simply floating around in the cytoplasm of the cell be able to reach a threshold for meaningful biological function? And if there are so many charged particles also floating around in the cytoplasm, at the same time, how could their influence not be blocked or interfered with? Doesn’t the cell need some level of thermodynamic stability to get anything done?

LLPS clarifies these concepts by identifying that inside cells, molecules that are charged come together and form “droplets”, or highly concentrated structures that, in a sense, amplifies the function of the individual molecule. Additionally, molecules exist in a more stable conformation when segregated into a condensed phase, compared to being in a diluted phase, where they are mixed in with other molecules. Meaning, molecules are “stronger” when they are together vs. when they are dispersed in a solution as individuals.

Luminescent molecular aggregates — example of pseudoisocyanine J-aggregates in aqueous solution. From Wikimedia Commons: original work by Oleksa.Sorokin

The aspect of LLPS that is applicable to biological function is that these segregated molecules continue to retain the structural flexibility of a liquid. Properties such as changes in size, and reversibility, allow the molecules in the aggregate to continue interactions and exchanges with the surrounding environment. This would not occur if this aggregate was inside a membrane-bound compartment. Essentially, LLPS creates functionally active structures that are membraneless, and these molecular aggregates concentrate in a particular area and actually create a sort of “macro-structure”. A colleague of mine explained that the structure can even resemble a sort of “rail” along which a signal can propagate.

Ultimately, LLPS is a simple, clean, and elegant explanation, bringing a sense of order to the chaos of the intracellular environment. The propensity of molecular aggregates to behave as organelles is an exciting venue for research in cell biology, and understanding the processes by which these aggregates form could also produce new therapies for disease conditions such as Alzheimer’s — a type of dementia characterized by the formation of protein aggregates.

I’m curious about the human experience | PhD in Neuroscience | Instagram: neuro_roksana

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