GENERAL ARTICLE An Introduction to Phase Separation in Cell Biology * Kripa Gowrishankar and Sravanti Uppaluri Phase transitions in cells provide a wonderful arena for in- vestigation by students of physics, chemistry, and biology to come together to apply their foundational knowledge. In this article, we provide an intuitive approach to the basic physi- cal principles of phase separation, We then highlight several examples that illustrate how the cell, the fundamental unit of life, makes use of phase separation to organise its contents. Principles of Intracellular Organisation One unifying principle that organises all life forms is that the cell is the fundamental unit of life. The cell membrane separates its contents from the environment. We lear in school that eukaryotic cells contain organelles like the nucleus, the endoplasmic reticulum, Golgi, etc., each with a specified function. We also learn that organelles are held together and separated from the cytoplasm by a lipid membrane. One thing we often miss while learning about these organelles is the highly dynamic nature of processes inside the cell. Students often think of the cell as a static entity, where every organelle has its fixed place and traffic is highly regulated and organised (Figure 1). In reality, it is busier than the busiest area of a metro city. Imagine a city with constant traffic in all directions and relentless entry and exit of materials in and out, Our cell would resemble this city, and the scenario just described would be business as usual for the cell. One of the advantages of this dynamic nature is that it enables the cell to adapt easily to any environmental changes, particularly environmental stresses. *Vol.28, No.2, DOI: hitps://doi.org/10.1007/s12045-023-1545-0 re" ‘Kripa Gowrishankar is an assistant professor of physics at Azim Premji University. Her research work is interdisciplinary, and she ‘uses tools from physics to understand biological processes. At APU she develops classroom demonstrations as well as projects that engage students as active learners and help build their intuition of physical phenomena. ‘Sravanti Uppaluri teaches biology at Azim Premji University. She studies developmental biology using Hydra and Celegans and works with undergraduate students to answer cell, size, and tissue organisation ‘questions. She is part of the author team of ‘wwwiThinkbiology.in. 229GENERAL ARTICLE Non—membrane bound —— sold fi Membrane bound dotted lve Nucleus Nucleolus Endoplasmic Reticulum Cajal Bodies Lysosome —_=—* Paraspeckles Hitochondria —S % Golgi body ——+—— %, % UG, Cell Membrane Figure 1. Organisation in- side the cell: Membrane- bound (labelled on the left) and non-membrane bound (labelled on the right) or- ganelles in the cell. Non- , and think of what would be happening inside the cell before and after the Imagine that a cell is exposed to environmental stres stimulus, Under pre-stress conditions, an important and ongo- ing process inside the cell is the production of messenger RNA membrane bound organelles (MRNA) molecules (transcription), getting ready to be translated are believed to assemble into proteins. When sudden stress is present, the cell needs to through phase separations. focus its energies on responding to it to protect its contents and potentially produce specific signals to respond to the stress. Keywords ‘To prevent the degradation and wastage of mRNA molecules al- Phase separation, membraneless yeady present in the cytoplasm during stress, mRNAs segregate organelles, RNP granules, demix. ing, cel. from the rest of the cell and into specific organelles (called stress granules) that seem to form spontaneously. Once the stress is removed, and the cell has recovered, these stress granules dis- assemble, and the mRNA is released [1] into the cytoplasm for future use. ‘What are stress granules made of? How do they assemble? It turns out that stress granules are part of a growing class of or- ganelles whose assembly and disassembly can be understood as liquid-liquid phase separation (LLPS). LLPS involves two liq- uids where one of the phases is a mixture, and another phase 230GENERAL ARTICLE is when a boundary appears between the two liquids, and they segregate from each other. This is analogous to another familiar phenomenon: a little oil in water quickly results in the formation of droplets and the two liquids, oil and water, ‘demix’. This hap- pens due to the properties of the molecules themselves, The oil molecules form droplets with hydrophobic cores to reduce inter- actions with water. If we were to increase the volume fraction of oil to water, we would see larger and larger oil droplets form until, eventually, we would see water droplets in oil. This change in the behaviour of the phases is illustrated further below. Something very similar happens inside the cell, Just as the oil | rust as the oil separates separates from water, a subset of protein and RNA molecules | from water, a subset of (or RNP bodies) ‘demix’ from the cytoplasm and form a new protein and RNA molecules (or RNP_ organelle, We call this organelle a stress granule as it forms in | jodies) demix’ from the response to external stress, cytoplasm and form a . . new organelle, We call However, such RNP bodies are not formed merely to cope with | this organelle a stress stressful conditions. They assemble for many other cellular fune- } granule as it forms in tions, such as ribosome production, gene expression control, cell] response to external division, and even embryogenesis—among a growing list of func- stress. tions being investigated. In addition to membrane-bound organelles such as the nucleus and mitochondria, phase-separated organelles also serve to com- partmentalise and organise the contents of a cell (Figure 1). Clearly, a basic understanding of phase separation will help to understand how cells function. In the following sections, we first introduce the reader to the mechanistic principles that drive phase separa- tion and then elaborate on some of the important functions of phase separation in cell biology. What is Phase Separation? Let’s consider a multi-component liquid system, like the cyto- plasm, containing a myriad of components, such as proteins, fats, and sugars. Ata high temperature (but not high enough to cause chemical changes), the different components freely move and col- lide fast enough so that no single type of component collects toGENERAL ARTICLE Phase separation can be understood using the broader framework of phase transitions, which includes several other physical phenomena form a distinct domain, However, as the temperature is brought down, the components move slowly enough to feel intercompo- nent interactions. This typically leads to the formation of distinct domains of one type of component, separated from the others by a boundary. This phenomenon is known as phase separation (also referred to as phase segregation, demixing). We shall see shortly that temperature is just one of the many external physical condi- tions that can trigger this phenomenon. Phase separation can be understood using the broader framework of phase transitions, which includes several other physical phe- nomena, Phase transitions came to be studied first in the physical transformations of matter from one form to another. The phase transitions exhibited by water (and other molecules like carbon dioxide, argon, nitrogen, ete.), changing from solid to liquid to ga ena that all of us have witnessed. Over the decades, the study of phase transitions has grown to include phenomena that aren’t just changes from solids to liquids to gases. The word phase refers to some physical attribute of the material that undergoes a change. For example, a common ferrite bar magnet is magnetic at room s the temperature or pressure changes, are familiar phenom- temperature but becomes non-magnetic (or paramagnetic) when sufficiently heated. The phase transition, in this case, refers to the transition from the magnetic to the non-magnetic phase as the temperature is raised. The material that makes up the bar mag- net remains solid through the transition. The phase of the mate- rial is captured instead by the magnetic strength of the material. Therefore, a phase can really refer to anything and depends on the transition we are witnessing. To understand phase separation in the cytoplasm, we can use what we know about phase transitions. The theory of phase transitions predicts that the system tends to a state with the lowest possible energy and highest possible entropy for a given set of external pa- rameters. In the following examples, we will illustrate the effect of these two factors separately. We will consider a much simpler system than the cytoplasm—a binary liquid (a mixture of two liq- 232 uids).GENERAL ARTICLE Some liquids, like alcohol and water, are miscible, meaning the molecules freely intermingle without discrimination. Alcohol and water can also form hydrogen bonds, which supports their ability to mix. If, however, enough salt is added to this mixture, the sol- vation of water by the ions of the salt makes water inaccessible to alcohol. The salty water can be seen to separate from the alco- hol, and a clear boundary can be seen between them. The binary liquid can be said to have undergone a phase transition from a well-mixed phase to a demixed phase [2]. In the above example, the strength of intermolecular attractive electrostatic interactions (that lower the energy) was tuned by the salt concentration, and the change in the interaction strength led to the observable effect of phase separation, Even if molecules have no chemical interactions, and physical collisions are the only interactions between molecules, phase sep- aration can occur due to a gain in the entropy of the demixed phase. Entropy is often thought of as a measure of disorder, and so a large entropy for a demixed phase may sound counterintu- itive. A more accurate way of understanding entropy is as fol- lows: A single molecule is free to diffuse, and its entropy is re- lated to the volume that is accessible to it. A system of many molecules would have several possible molecular arrangements, and the entropy of the combined system is related to the number of such arrangements. For example, let's consider two identical molecules where each can occupy one of the four positions. If the molecules are not allowed to overlap, then the number of ar- rangements is *C2, and the entropy is ~log(#C2). Increasing the number of positions increases the entropy of this system, To understand how entropy drives phase separation, we consider a binary liquid with two types of particles, A and B, of radii Ra and Rg. Particle A is much larger than B (Ra >> Ry). Both particles are rigid and cannot overlap in space. Consider a single particle of type A, like in Figure 2. Particles of type B cannot access the volume occupied by A and an exclusion zone of thickness Ry (shaded region) around it. If particles of type A come together, as seen in the right panel, we see that the exclusion zone is smaller. Even if molecules have no chemical interactions, and physical collisions are the only interactions between molecules, phase separation can occur due to a gain in the entropy of the demixed phase 233GENERAL ARTICLE Figure 2. Phase separation in a two component system driven by entropy. ‘The phase diagram is a graph where all externally tuneable parameters are scanned on the axes and boundaries at which phase changes happen are drawn, As aresult, particle B has access to more free volume This means that the segregated phase (right panel) has a higher net entropy than the mixed phase (left panel), To summarize, both energy and entropy need to be considered while investigating phase separation. In the example of water and ethanol, energy was driving the phase separation, while in the second example (Figure 2), it was entropy. ‘What can you control externally to make the transition happen? Often, temperature is an important driving force. There can be others, like pressure, magnetic field, pH, salt concentration, ete. These parameters are typically systematically tuned in a lab while also observing the phase of the system, and this is represented pictorially in a ‘phase diagram’. The phase diagram is a graph where all externally tuneable parameters are scanned on the axes (there can be more than two axes, but that's difficult to draw) and boundaries at which phase changes happen are drawn, as shown in Figure 3 The framework of phase transitions discussed above is valid for all systems—living and non-living. However, phase separation can also be triggered by processes unique to the living world. The cell is, as we said earlier, like a city with heavy traffic. The movement inside the cell is not just a result of diffusion but isGENERAL ARTICLE (salt, pH, temperature etc) Interaction parameter actively generated and requires fuel in the form of ATP. Molec- ular motors, actin filaments and microtubules make up the strue- tural machinery that enables the transport of materials. In such a milieu, phase separation can be triggered by ‘active’ (or energy- consuming) processes. The uncovering of active mechanisms at play inside the cell is very much a topic of current research. Phase Separation in Biology How Is Phase Separation Also Important for Living Things? In cells, there are a large number of different circumstances that can change the interactions between molecules and induce phase separation. These include changes in salt concentration, change of pH, presence of RNA or protein abnormalities etc., which can cause a demixing transition and eventually result in the formation of a new membrane-less organelle. Non-membrane bound RNP granules have many essential func- tions, Therefore, the mechanism that drives their assembly and disassembly and properties have been the subject of many recent studies. The studies probe questions such as: ‘© What types of proteins assemble into these organelles? ‘* Are there any general features of the protein that makes it more volume fraction of oil in water Figure 3. Phase diagrams the phase diagram for two immiscible fluids such as a mixture of oil and water, Changes in salt concentration, change of pH, presence of RNA or protein abnormalities etc., can cause a demixing transition and eventually result in the formation of a new membrane-less organelle, 235GENERAL ARTICLE Early work showed that low complexity multivalency, that is, repeats of short sequences of amino acids, enhanced a protein’s ability to phase separate, likely to assemble into these organelles, such as amino acid com- position, charge, size, etc.” ‘© How does assembly change in the presence of a mutation? ‘© What is the role of RNA in demixing? ‘© What are the physical properties of the phases, such as viscosity, and how do they influence the function? * Finally, what kinds of functions do RNP granules serve? What Are the Properties of the Phase Separating Molecules In- side the Cell? Recent work has only just begun to uncover the details of the composition of RNP granules in the cell. However, several pat- tems in terms of the amino acid composition and structure of component proteins have been identified and are suspected to fa- cilitate phase separation, Early work showed that low complexity multivalency, that is, re- peats of short sequences of amino acids, enhanced a protein's ability to phase separate. For instance, relatively large portions of the proteins in RNPs are repeats of arginine and glutamine (RGG repeats). Repeats of proline-rich domains have also been shown to phase separate in vitro [3]. Interestingly, RGG sequences have long been classified as RNA binding sequences (also called motifs) — clearly highlighting the importance of the interaction between RNA and protein in these phase-separated bodies. In addition, hydrophobic domains naturally lend themselves to phase separa- tion, as we saw in the oil droplets described earlier. The presence of these kinds of sequences in a protein could also lead to intrin- sically disordered structures (see Figure 4 for an illustration of disorder in protein structures), which means that the protein lacks a stable and locked-in 3D structure. Indeed, this is seen to be a very common feature of proteins found in RNP granules, To understand what sets off the formation of RNP granul need to understand the molecular details of component molecules 236GENERAL ARTICLE secondary se I disorder -nanna-m=— structure and the interactions between them. This is what allows them to overcome entropy and demix from the cytoplasm or nucleoplasm. ‘These studies are being conducted both in vivo, by observing fluorescently tagged protein localization, and in vitro, by isolat- ing particular proteins and observing their phase behaviour under varying conditions of temperature, pH, salt, etc, Notably, the na- ture of interactions between RNP components may determine the biophysical characteristics (ranging from viscous liquids to gel- like) as well as functional properties. Functions ‘The protein/RNA granules that phase separate usually serve as biochemical reactors. These reactors facilitate interaction between their component molecules and thus serve specific functions based on their composition, Examples of such functions include ribo- some biogenesis in the nucleolus, mRNA turnover in P-bodies, and telomere assembly in Cajal bodies (see Figure 1). Gene Expression One of the most fascinating aspects of developmental biology is the process of cell differentiation: how a single-celled embryo divides, leading to different cell types. The basis of cell differen- tiation is differential gene expression; expressing different parts Figure 4. Differences between a disordered pro- tein and a structured pro- tein. The disordered protein does not take on a fixed 3- dimensional shape. The proteinfRNA granules that phase separate usually serve as biochemical reactors. ‘These reactors facilitate interaction between their component molecules and thus serve specific functions based on their composition. 237GENERAL ARTICLE Phase separation helps determine which genes are turned ‘on’ or ‘off ‘This, in turn, can lead to cell differentiation and is important for development. Thus, phase separation plays a vital role in biological processes. of the same genome in varying combinations can give rise to dif- ferent cell types. The transcription of RNA of protein-coding genes from corre- sponding DNA sequences within the nucleus can be broken down into three processes: transcription initiation, elongation, and ter- mination. All three processes require a larger number of proteins to be recruited to a specific location on the genome. For exam- ple, transcriptional initiation begins at a site right at the start of the gene called the promoter. Transcription is catalysed by an en- zyme, RNA polymerase II (Pol-ID). For Pol-II to bind to the pro- moter site, various proteins called transcription factors (TFs) are required, The nucleus is a densely packed compartment. There- fore, when a gene has to be expressed, the location of the tran- scriptional machinery has to be precisely controlled, There is mounting evidence that many TFs have disordered protein do- mains that help form phase-separated bodies, also called conden- sates, around Pol-I. A demixing transition can be induced in ar- eas where they are required to form condensates. This mecha- nism, therefore, helps concentrate and properly locate TFs wher- ever they are needed. Another advantage of this mechanism is that additional condensates can be formed around the elongation complex. This process allows the initiation and elongation com- plexes (both of which share Pol Il, but not all TFs) to be seg- regated from each other and so is thought to result in tight spa- tiotemporal control [4] (see Figure 5). Therefore, phase separa- tion helps determine which genes are tumed ‘on’ or ‘off”. This, in tum, can lead to cell differentiation and is important for devel- opment. Thus, phase separation plays a vital role in biological processes. Ribosome Biogenesis in the Nucleolus The nucleolus is often depicted in cell biology textbooks as the largest structure in the nucleus. It is actually a manufacturing site for ribosomes (also called ribosome biogenesis). The nucle olus assembles and disassembles during cell division. In many 238GENERAL ARTICLE cell types, in the initial phases of assembly, many small nucleoli are seen, but over time their number decreases, but their size in- creases. How can this behaviour be explained? It turns out that nucleoli also assemble via demixing or phase separation. The in- crease in size (and the corresponding decrease in number) could be attributed to the fusion of nucleolar phases—much like two oil droplets fusing into one large droplet when they come into contact while in water. In fact, Brangwynne et al, showed that nucleoli display liquid- like behaviours: droplet fusion, rounding to a spherical shape (to minimise energetic costs), and a characteristic viscosity [5]. The nucleolus is especially interesting because it is composed of at least three different immiscible phases. This means that the nu- cleolus contains coexisting liquid phases, which form subcom- partments. This organisation mediates the functions of the nucle- olus. The innermost layer, the fibrillar centre (FC), is where RNA polymerase I (Pol-I) is active; the middle layer, the dense fibrillar Figure 5, Condensates or phase-separated mixtures of transcription factors and RNA polymerase (black ovals) keep transcriptional machinery segregated for different processes, such as termination and elongation. ‘The nucleolus is, especially interesting because it is composed of at least three different immiscible phases. 239GENERAL ARTICLE The nucleolus is an incredible example of a non-membrane bound organelle whose functions are facilitated by the simple physical principles of surface tension leading to coexisting liquid phases. Figure 6. Demixed com- ponents of the nucleolus (which is phase-separated from the nucleoplasm) come together to form the or ganelle. Because of the immiscibility of the com- ponent proteins, the nu- cleolus forms a multicom- ponent phase-separated or ganelle. This is an essential feature of the function of the nucleolus, component (DFC), is where rRNA is processed; and the outer- most layer, the granular component (GC), is where ribosome bio- genesis is completed (see Figure 6). When conditions are right, each component is immiscible with the other and the surrounding cytoplasm. This means that all components would tend to demix from each other, and we could expect to see regions containing only one component, separated from the others at interfaces (cor- responding to smaller, more numerous ‘nucleoli’). The forma- tion of the interface between two liquids is governed by attractive forces between molecules at the interface (known as interfacial tension). The peculiar structure of the nucleolus can be under- stood by comparing the interfacial surface tensions between the different pairs of liquids [6]. This is an incredible example of a non-membrane bound organelle whose functions are facilitated by the simple physical principles of surface tension leading to co- existing liquid phases. (multiphase organelle) Viral Replication Itis not only mammalian cells that leverage the potential of LLPS as a tool to spatially and chemically sequester proteins and RNA. Recent work has demonstrated that both human respiratory syn-GENERAL ARTICLE Box 1. Exercise Let’s consider two immiscible liquids, A and B, which are immersed in a medium made up of another liquid, C. Any interface between pairs of these liquids will have an interfacial tension, and we denote these by op, onc, oc. The energy of this system is determined by the perimeter of the interfaces between the different liquids. In this particular case, we assume that the particles ate point objects and so we do not consider any change in entropy due to excluded volume effects. If pAB, pBC and pAC are the perimeters of the interfaces, then the energy of this system is E = oagpAB + opcpBC + ocpAC Now consider the three configurations shown in Figure (a). In all three panels, component A occupies as much area as component B, For example, in panel 1, the radii of the two separate domains of A and B are both the same and equal to r. In panel 2, component A gets redistributed as a ring around component B, and the radius of the combined circle (inner circle + ring) is R. Which configuration do you think the system will ake on, given the following values of the interfacial tensions: ora = rac = | unit of energy/length and xc = 0.4 units of energy/length. What would happen if oan > onc? 1. 2 3. Figure (a): Which configuration is the most likely, given the values for surface tension provided? (Hint: all you have to do is calculate the perimeters of the interfaces and use the formula for energy) cytial virus (RSV) and SARS-CoV-2 utilise LPS for viral RNA synthesis and targeting innate host immunity, respectively. RSV infections induce the formation of viral inclusion bodies (IBs) that have liquid phase properties inside the host cell. IBs are the primary site of viral RNA synthesis and hence are essen- tial for viral replication. The inclusion bodies can be disrupted in two ways; either by dissolving the liquid phase (moving into the L-phase regime of the phase diagram, see Figure 2) or by hard- ening the IBs to have solid-like properties. Both these methods could be used as targeted therapies against RSV. Indeed, Risso- Inclusion bodies are the primary site of viral RNA synthesis and hence are essential for viral replication. TesoNANCE Fy “Ww 2aGENERAL ARTICLE Figure 7. When the RSV virus infects a host cell, it releases viral RNA. This vi- ral RNA is found in inclu- sion bodies, which are also RNP phases. Hardening this phase (removing its liq- uid properties) prevents viral RNAs from being released for further processing and can, therefore, interfere with the pathogen. 1s particular amino acid se quence on the protein that al lows the protein to form dimers. _— Hose colt Ballester et al. [7] found a drug (alkaloid cyclopamine) that could harden the IBs. Hardening the IB liquid phase abolished viral replication, as seen in Figure 7. ‘The hallmark of many neurodegenerative diseases is actually the accumulation of protein aggregates, some which are now believed to arise from RNP proteins. In fact, Amyotrophic lateral sclero- sis (ALS), is associated with mutations in FUS, a protein which normally assembles as liquid droplets at sites of DNA damage. In ALS patients, the mutations result in irreversible aggregation of protein; similar to the hardening of viral inclusion bodies de- scribed above, The irreversibility of this process could play a major role in disease onset and progression of ALS. Many neu- rodegenerative diseases, not just ALS, are associated with such protein aggregates, and studies that identify irreversible protein aggregation may shed light on disease mechanisms, In another study, Wang et al. showed that demixing of the SARS- CoV-2 nucleocapsid protein (SARS2-NP) from the cytoplasm me- diated innate immune evasion from the host. They found that the dimerization domain! is required for the phase separation of m2GENERAL ARTICLE SARS-NP. The viral RNA is protected within the liquid phase of SARS-NP bodies. Targeting the dimerization domain of SARS- NP disrupted phase separation and made viral RNA more suscep- tible to host immune response [8] These two studies revealed the importance of liquid phase separa- tion in viral replication and suppression of host immune response and also pointed to paradigms for therapeutic targets. Aberrant Phase Separation, Ageing and Neurodegeneration One of the most interesting features of phase separation in biol- ogy is its reversibility. The cell can tune the values of the interac- tion parameter to switch between mixed (cytoplasm) and demixed (cytoplasm with distinct organelles) phases. The reversible nature of phase separation allows cells to respond to a dynamic environ- ment (as seen in Figure 8) The hallmark of many neurodegenerative diseases is the accu- mulation of protein aggregates, some of which are now believed to arise from RNP proteins. In fact, amyotrophic lateral sclero- (ALS) is associated with mutations in FUS, a protein which normally assembles as liquid droplets at sites of DNA damage. In ALS patients, the mutations result in irreversible aggregation of the protein, similar to the hardening of viral inclusion bodies described above. The irreversibility of this process could play a major role in disease onset and progression of ALS. Many neu- rodegenerative diseases, not just ALS, are associated with such protein aggregates, and studies that identify irreversible protein sis Figure 8. The physi- cal properties of FUS pro- tein phases may change with ageing. These are hall- marks of neurodegenerative disease. One of the most interesting features of phase separation in biology is its reversibility, ‘The hallmark of many neurodegenerative diseases is the accumulation of protein aggregates, some of which are now believed to arise from RNP proteins, TesoNANCE Fy “Ww 243GENERAL ARTICLE aggregation may shed light on disease mechanisms [9] Conclusion This was just a bird’s eye view of phase separation and how and when it takes place inside the cell, We learnt that phase transi- tions are driven by a balance between energy and entropy. In the cell, such transitions occur as liquid phases separating or demix- ing from each other. This kind of liquid-liquid phase separation is essential for fundamental biological processes ranging from gene expression to ageing. Gaining an understanding of these phase separations in biology will give us insights into the inner workings of the cell—how it is organised (i.e., how functions are segregated into organelles), how this organisation affects function, and ultimately what hap- pens when things go wrong in disease. Recognising that phase transitions take place in a cell could only be done through an inter- disciplinary approach—leveraging foundations in physics, chem- istry, and biology! We hope this article encourages you to look at biology as an interdisci Suggested Reading [1] A Molliex et al, Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization, Cell, Vol.163, pp.123- =133, 2015. [2] Svenja Lohner, Separate Liquids with Salt!, Scientific American, btps: //mmw.scientificamerican.con/article/separate-Liquids-with-salt/. [3] P Lict al, Phase transitions in the assembly of multivalent signalling pro- teins, Nature, Vol.483, pp.336-340, 2012. [4] P Cramer, Organization and regulation of gene transcription, Nature, ‘Vol.573, pp.45-54, 2019. [5] © P Brangwynne, T J Mitehison and A A Hyman, Active liguid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes, PNAS, 108 (11), pp-43344339, 2011. https: //doi .org/10, 1973/pnas. 101715010. [6] M Feric et al,, Coexisting liquid phases underlie nucleolar subcompartments, Call, Vol65, pp.1686—1697, 2016. [7] J Risso-Ballester et al., A condensate-hard in vivo, Nature, VoLS9S, pp.596—599, 2021. 1g drug blocks RSV replication il RESONANCE | February 2023GENERAL ARTICLE [8] S Wang et al., Targeting liquid-liquid phase separation of SARS-CoV-2.nu- cleocapsid protein promotes innate antiviral immunity by elevating MAVS Nat Cell. Biol, Vol.23, pp.718—732, 2021, [9] A Patel et al, A liquid-to-solid phase transition of the ALS protein FUS ‘Address for Correspondence celerated by disease mutation, Cell, Vl.162, pp.1066—1077, 2015. Kripa Gowrishankar Email kripa.gowrishankarsapu edu.in Sravanti Uppaluri Email sravanti -uppaluri@apu edu.in ‘Azim Premji University Burugunte Village Survey No 66 Bikkanahalli Main Ra Sarjapura Bengaluru, Karnataka 562 125,