- Open Access
Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation?
© Iborra; licensee BioMed Central Ltd. 2007
Received: 27 October 2006
Accepted: 12 April 2007
Published: 12 April 2007
The cell nucleus is highly compartmentalized with well-defined domains, it is not well understood how this nuclear order is maintained. Many scientists are fascinated by the different set of structures observed in the nucleus to attribute functions to them. In order to distinguish functional compartments from non-functional aggregates, I believe is important to investigate the biophysical nature of nuclear organisation.
The various nuclear compartments can be divided broadly as chromatin or protein and/or RNA based, and they have very different dynamic properties. The chromatin compartment displays a slow, constrained diffusional motion. On the other hand, the protein/RNA compartment is very dynamic. Physical systems with dynamical asymmetry go to viscoelastic phase separation. This phase separation phenomenon leads to the formation of a long-lived interaction network of slow components (chromatin) scattered within domains rich in fast components (protein/RNA). Moreover, the nucleus is packed with macromolecules in the order of 300 mg/ml. This high concentration of macromolecules produces volume exclusion effects that enhance attractive interactions between macromolecules, known as macromolecular crowding, which favours the formation of compartments. In this paper I hypothesise that nuclear compartmentalization can be explained by viscoelastic phase separation of the dynamically different nuclear components, in combination with macromolecular crowding and the properties of colloidal particles.
I demonstrate that nuclear structure can satisfy the predictions of this hypothesis. I discuss the functional implications of this phenomenon.
The cell exist a crowded environment of organelles, macromolecules, chromatin, membranes, and cytoskeletal filaments. The cell is not, however, simply a soup of its constituent parts, rather there exists an ordered structure referred to as compartmentalisation. Maintenance of compartmentalisation within the cell has fundamental implications for cellular function. In the cytoplasm, compartmentalisation is commonly achieved by confining macromolecules in lipid membranes thereby creating organelles such as mitochondria, lysosomes, Golgi apparatus, etc. However, even the cytoplasm regions not divided by membranes can show local differences in composition. Within the nucleus there also exist numerous distinct structures such as the nucleolus, interchromatin granule clusters (IGC), heterochromatin, and various bodies such as: Cajal, PML, SMN. Nuclear compartmentalization exists without any membranous division. Key questions such as how nuclear compartmentalization is achieved and why it exists, still remain unanswered. In a seminal paper Tom Misteli proposed self-organization as an explanation for the existence of nuclear compartmentalization  but the molecular basis for self-organization of nuclear structures is not fully understood. Another phenomenon implicated in nuclear compartment formation is macromolecular crowding, however, this only explains the existence of some of the nuclear structures , but is not enough to explain the different structures found in the cell nucleus. Several models have been proposed to explain three-dimensional chromatin organization, from modelling chromatin as balls connected by springs [3–5] to chromatin loops as semi flexible (self-avoiding) tubes . All these models are very simplistic, tending to focus on chromatin as an independent entity floating in an ideal buffer. No consideration is given to the physical properties of the nuclear components and its consequences for nuclear structure. The main stumble block to date is no one model can fully account for the diversity of nuclear structures observed. Recent advances in biophysics have provided us with invaluable information and have allowed us to understand cell organization. In this paper I explore a biophysical explanation for compartmentalization within the cell nucleus.
Dynamic Asymmetry within the Nucleus
Nuclear DNA is associated with histones, which are then packaged into an ordered structure called chromatin. This chromatin is further packaged into individual chromosomes that occupy distinct territories in the nucleus . Within the mammalian nucleus, chromosomes territories show non-random, evolutionarily conserved radial organisation on the basis of gene content. Gene-rich chromosomes occupy a more internal nuclear location and gene-poor chromosomes reside at the nuclear periphery [8–10], which may be driven by the interaction of heterochromatin with the nuclear lamina . While chromosome territories are more or less fixed throughout the cell cycle except for early in G1  their constituent chromatin does show a degree of constrained diffusional motion. Chromatin dynamics in living cells have been studied by several groups by exploiting the lac operator/repressor system . Integration of a lac operator array into the DNA of cells expressing GFP-lac repressor fusion protein allows chromatin movement to be monitored. The main findings of these studies are that chromatin moves in a Brownian manner with a diffusion coefficient in the range ~10-4 to 10-3 μm2/s [12, 14]. Chromatin mobility is also affected by condensation state; euchromatin moves faster than heterochromatin .
Nuclear protein dynamics have also been studied extensively using photobleaching experiments, namely fluorescence recovery after photobleaching (FRAP), and fluorescence loss in photobleaching (FLIP). Experimental evidence shows that proteins are highly dynamic and move unrestricted through the nuclear volume in an energy-independent manner . Whilst roving through the nuclear space a protein may engage with non-specific or high-affinity binding sites, as demonstrated by Phair et al  who estimate residence times of 2–30 s for chromatin proteins on both euchromatin and heterochromatin, and the time between binding events at around 100 ms. Some times diffusion is so rapid that FRAP or FLIP approaches are not suitable to calculate the diffusion coefficient. In these instances, diffusion measurements are performed using fluorescence correlation spectroscopy (FCS) . This technique allows one to calculate the average time needed for a fluorescently labeled molecule to pass through a very small defined confocal volume. Since the confocal volume is a known measure the diffusion coefficient (D) of the molecule can be determined. Freely diffusible proteins within the nucleus move slower than in water but the D values are in the 0.2 to 20 μm2/s range [1, 18]. In addition to proteins exhibiting highly dynamic diffusion, nuclear bodies also show a degree of motion. Gorisch et al. have studied the diffusion properties of both Cajal and PML bodies as well as a biochemically inactive body composed of murine Mx1 . Their findings indicate that nuclear bodies show constrained diffusion within a chromatin corral, which can itself translocate.
The movement of RNA also can be measured by Fluorescent RNA Cytochemistry [20, 21]. Photoactivation of caged fluorochromes conjugated to oligonucleotides allow of RNA molecules visualisation in living cells. In living cells the RNA demonstrates Brownian motion, with diffusion constants ranging from 0.1 to 10 μm2/s [20, 22].
These observations show a nucleus with two levels of dynamics; a slow chromatin compartment and a fast compartment of proteins and RNA.
Dynamic asymmetry leads to viscoelastic phase separation
Crowding effects have been demonstrated to play an important role in formation of some protein-based nuclear compartments. It has been shown that nucleoli and PML bodies disassemble when nuclei are expanded to twice their normal volume by hypotonic treatment . Reassembly of these compartments can be achieved by either returning the nuclear volume to normal in standard buffer or, alternatively, by adding inert macromolecules to expanded nuclei. In addition, the dynamic nature of compartments, with a constant exchange of macromolecules between the compartment and the nucleoplasm, is consistent with the properties of demixed phases produced by crowding . The spherical/spheroid shape of nuclear bodies are typical morphologies generated by macromolecular crowding, as this globular conformation is favoured by macromolecular crowding theory [36, 37]. Nevertheless, macromolecular crowding in itself is not enough to generate compartments. We know that the expression of GFP alone does not generate any kind of structure arguing for the need for some kind of self-associating properties in the constituents of the body. This has been shown to be the case in the well-studied Cajal body, where the presence of self-interacting proteins is required for the body formation and also in PML or SMN bodies [40–43]. Recently it has been shown that SUMO modification of PML components is essential for PML body formation. It seems that proteins containing SUMO-binding motifs act as a scaffold for the formation of large macromolecular complexes [44, 45]. Interestingly, assigning specific functional roles to these two bodies in particular has been elusive.
It may be that these bodies are the result of pure biophysical forces and have no truly functional role as a body. In this vein, we know that chemical reactions are a function of interaction between molecules, which interact through their surfaces. Reactivity can be viewed as a problem of molecular surfaces. The surface area associated with a given mass of material subdivided into equal-size particles increases in inverse proportion to the linear dimensions of the particles. Put simply, the bigger the structure, the lower the surface area exposed. This point can be easily illustrated by example of a small molecule of 10 nm of diameter that can self-interact, building structures from 200 nm to 1 μm. Using a 10 nm monomer, 8000 and 106 molecules are respectively required to build a 200 nm or 1 μm structure. The reduction in surface of these particles in the bulk state is strikingly evident; 95 and 99% reduction, in the case of 200 nm and 1 μm respectively. The argument is obvious; when proteins accumulate in bodies their specific activity (Units of activity per molecule) become reduced by a factor proportional to the size of the body. This would support the premise that nuclear bodies are less likely to represent a functional state.
Colloidal properties: The speckle compartment as an example
A distinctive compartment, which illustrates another type of physical force operating in the nucleus, is the IGC. Within mammalian cells, pre-messenger RNA splicing machinery is found in a compartment referred to as the speckles, splicing factor compartment, SC-35 domains or IGC. By fluorescence microscopy these nuclear speckles are seen as irregular shaped bodies located at interchromatin regions. When nuclear speckles are examined by electron microscopy, they can be seen to be composed of clusters of interchromatin granules, measuring 20–25 nm in diameter . They contain numerous factors involved in RNA synthesis and processing and seem to be involved in assembly or modification of these factors [46, 47]. Unlike other nuclear bodies, the nuclear speckle compartment is known to be positionally stable. Time-lapse observations of nuclear speckles in living cells have shown that their position is maintained over many hours [48, 49].
IGC is a reversible flocculate of colloidal particles and as such structure is irregular and bulky, which makes it practically immobile, in perfect agreement with the documented possitional stability of IGCs .
The predominant force determining structure in the case of nuclear speckles seems to be the colloidal properties of interchromatin granules.
The nuclear matrix: another manifestation of colloidal properties of nuclear constituents
A debatable compartment is the nuclear matrix. This compartment is visible after extensive extraction using detergents, high salt solutions and treatment with nucleases. The nuclear matrix is a fibro-granular network with a complex protein composition [54, 55]. Many scientists have tried to visualize this structure in un-extracted cells by light or electron microscopy, but these attempts have been unsuccessful. They have tried to express these proteins tagged with GFP or using specific antibodies to visualize the nuclear matrix, but all these efforts have failed to demonstrate the existence of nuclear matrix. For these reasons many scientists believe that the nuclear matrix is no more that an experimental artifact that has nothing to do with the physiology of the cell. Moreover, many of the proteins implicated in the nuclear matrix formation are known to be highly dynamic which would appear to be in direct opposition of the idea of a nuclear skeleton.
I believe that some important clues to the solution of this controversy lie in the physical properties of macromolecules. Almost every single macromolecule found in the cell nucleus is a colloid (colloids have sizes ranging from 1 nm to 1 μm). Colloidal particles have a very distinctive set of properties. Their solubility properties strongly depend on the ionic conditions of the medium; just changing the ionic conditions means that the solubility of colloids changes. Colloidal particles in solution carry a electric charge, which have dual origin: the pH and ionic composition of the medium . The pH will determine the ionization of the radical groups of the amino-acid chains (in the case of proteins) or the phosphate groups (in the case of RNA). The ions present in the medium will be adsorbed on to the surface of the colloidal particle and together with the pH will determine the solubility properties of the particle under physiological conditions. We know the range of physiological pH inside the nucleus, but we are far from an exhaustive and detailed knowledge of the ionic composition. The coagulation of colloidal particles is very well known for more than a century – Faraday in 1856 described the coagulation of colloidal particles by addition of ions to the media- . Therefore one could imagine that nuclear matrices are in fact aggregations of colloidal particles, which precipitate giving the beautiful structures seen in nuclear matrix preparations. In this way when ribonucleoproteins hnRNP A2 and hnRNP B1, at low protein concentration, are exposed to high ionic strength buffers, they associate in regular helical filaments ranging in length from 100 nm to 10 μm with diameters from 7 to 18 nm. However, when the protein concentration was raised, the filaments rapidly aggregated forming thicker filamentous networks that look like the fibrogranular structures of nuclear matrices . For these reasons it is not surprising to find MARs (Matrix Attachment Regions) in nuclear matrix preparations, because these MARs are transcriptionally active, therefore the RNA bound to RNPs emerging from them will aggregate in the artefactual nuclear matrix preparation, dragging the RNA pol II associated with the DNA (MARs).
Functional implications of viscoelastic phase separation and macromolecular crowding
Understanding the mechanisms of compartmentalization is essential to understanding nuclear processes and their control. By separating the non-specific biophysical effects of phase separation and macromolecular crowding from the truly specific interactions, one can really begin to appreciate nuclear organization and its relation to function. Spherical nuclear bodies can be explained by macromolecular crowding effects, but not the sponge-like structure adopted by chromatin, which is explained by viscoelastic phase separation. If one accepts that nuclear components are subject to viscoelastic phase separation, the implications of such a phenomenon can be addressed.
If phase separation does occur in the living cell then interactions between the phases, namely the chromatin and protein compartments, should be limited to the interface between the two. This is indeed what we observe. It is well known that colloidal particles (eg. proteins, spliceosomes, RNPs) partition at the interface in systems where phase separation occurs [57, 58] – this will be enhanced in proteins with an affinity for DNA. Proteins adsorbed at the interface would exhibit reduced Brownian movement. This could be an alternative interpretation to models that propose chromatin associated proteins first associate with nonspecific sequences of DNA at low affinity and then diffuse along the DNA in one dimension searching for a "bona fide" binding site . Nonspecific binding of these proteins to the DNA could account for the population demonstrating intermediate dynamics between freely diffusible and fully bound .
In conclusion, I propose the hypothesis that nuclear compartmentalization is the result of the physical forces operating in the cell nucleus. In this way the different structures observed will depend on the characteristics of the compartment components. When viscoelastic phase separation is the prominent force, the compartment generated will have sponge like structure. When no strong dynamic asymmetry applies to the components of the compartment and self-association occurs, then macromolecular crowding is the driving force generating spherical structures. If no dynamic asymmetry applies to the components of the compartment and they do not self-associate, then no phase separation will operate and the compartment will look unstructured. If colloidal properties are predominant, the structure will behave like a colloidal gel.
The concepts of viscoelastic separation and macromolecular crowding appear to be consistent with the literature in the field of nuclear structure and function. Appreciation that biophysics has an important role to play in nuclear organisation will doubtless lead to a better understanding of the functions of the nucleus and more importantly give insight what may be happening when these processes go awry.
This work was supported by the MRC. Thanks to T Hernandez for help and J Brown and V Buckle for discussion and help. I dedicate this work to my family for understanding and support, especially to my father.
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