Available online at www.sciencedirect.com ScienceDirect Nuclear bodies: the emerging biophysics of nucleoplasmic phases Lian Zhu and Clifford P Brangwynne The cell nucleus contains a large number of membrane-less take four times as long. Diffusive transport could thus be a bodies that play important roles in the spatiotemporal rate-limiting step for many nuclear processes, particularly regulation of gene expression. Recent work suggests that low those involving a large number of binding partners. complexity/disordered protein motifs and repetitive binding ‘Active diffusion’, driven by non-equilibrium, ATP-de- domains drive assembly of droplets of nuclear RNA/protein by pendent dynamics, could partially address this problem promoting nucleoplasmic phase separation. Nucleation and by speeding up dynamics within the nucleus [1,2]. The maturation of these structures is regulated by, and may in turn three dimensional architecture of the genome itself is affect, factors including post-translational modifications, likely important, controlling the spatial proximity of co- protein concentration, transcriptional activity, and chromatin regulated genes and enhancer elements [3,4]. However, state. Here we present a concise review of these exciting recent establishing efficient transport and reaction rates remains advances, and discuss current and future challenges in a potential challenge, particularly given the densely understanding the assembly, regulation, and function of crowded nature of the nucleus [5]. nuclear RNA/protein bodies. Address Non-membrane bound nuclear bodies may play an impor- Princeton University, Department of Chemical and Biological tant role in addressing this problem by spatially patterning Engineering, Princeton, NJ 08544, USA molecular concentration in the nucleoplasm. Indeed, these structures locally increase the concentrations of molecules Corresponding author: Brangwynne, Clifford P involved in chromatin remodeling, transcription initiation, ([email protected]) and RNA processing. These structures could also play a role in functioning as biophysical switches, responsive to Current Opinion in Cell Biology 2015, 34:23–30 local signals by assembling only above a threshold concen- tration. Understanding the biophysical rules governing This review comes from a themed issue on Cell nucleus their assembly and properties thus promises to shed light Edited by Karsten Weis and Katherine L Wilson on the important and still poorly understood aspects of gene regulation in the nucleus. http://dx.doi.org/10.1016/j.ceb.2015.04.003 Nuclear bodies 0955-0674/# 2015 Published by Elsevier Ltd. The problems of transport and molecular reaction rates in the nucleus are similar to those encountered in the cytoplasm, where organelles play a central role in orga- nizing the spatial and temporal distribution of molecules. Large organelles are sometimes even further sub-com- partmentalized, such as in the cristae of mitochondria. It Introduction is thus not surprising that as the largest organelle in the Understanding the structure and function of the nucleus cell, the nucleus contains a variety of sub-compartments is essential for deciphering the computational logic em- to organize the nucleoplasm. bedded in the genome. It is increasingly apparent that there is an intimate connection between the 3D structure The sub-organelles of the nucleus are membrane-less, within the nucleus and biological function. However, the making their assembly and regulation particularly inter- strongly non-uniform spatial distribution of molecules esting. These structures are referred to as RNA Granules, within the nucleus is often neglected, even while it is RNP bodies, or recently RNP droplets; here we will refer clear that these variations could have a large impact on the to them simply as nuclear bodies. Examples include Cajal molecular interactions governing gene regulation. bodies (CBs), speckles, Histone Locus Bodies (HLBs), PML bodies, and nucleoli — the largest and prototypical Concentration variations within the nucleus are likely to nuclear body (Figure 1); numerous RNP bodies can also be important due to their effect on reaction rates and be found in the cytoplasm. Although often exhibiting diffusive transport. Diffusion is a stochastic, non-direc- cell-cycle dependent assembly and disassembly, these tional process, and thus diffusive transport rates depend structures can remain stable over timescales of minutes to not on the distance (as for directed transport), but on the hours, even while their components are in a constant state distance-squared — for example, to go twice as far it will of dynamic flux with the surrounding nucleoplasm [6]. By www.sciencedirect.com Current Opinion in Cell Biology 2015, 34:23–30 24 Cell nucleus Figure 1 10 μm DIC DAPI Synthetic Bodies Nucleoli PML Bodies Merge Current Opinion in Cell Biology Examples of nuclear bodies. Examples of nucleoli, PML bodies, and synthetic Ddx4YFP bodies assembled in HeLa cells. DNA is stained with DAPI. Adapted with permission from [17 ]. concentrating molecules within a small micro-compart- vice versa. These phase changes can be a strong function ment of the nucleus, while allowing these molecules to of salt, protein concentration, and temperature, parame- remain dynamic and mobile, these structures may func- ters that affect the free energy governing interactions tion to increase reaction rates [7] much like conventional between proteins. The hypothesis that phase separation membrane-bound cytoplasmic organelles. underlies nuclear body assembly is attractive because it provides a well-established physical framework for un- Elucidating biophysical principles underlying nuclear derstanding how the interactions of large numbers of body assembly is a formidable challenge, particularly molecules could manifest in their spontaneous, non-se- given the large number of components and dynamic quential self-assembly. nature of these assemblies. Moreover, research over the last decade suggests that nuclear bodies can be nucleated Consistent with the phase separation hypothesis, nuclear by a variety of components [8,9]. This presents an addi- bodies often behave like liquid droplets of RNA and tional conceptual difficulty, since nuclear body assembly protein. For example, it is well known that somatic cell may be largely ‘non-sequential’, which contrasts with the nucleoli often fuse with one another. Using the large and ordered, sequential assembly steps typical of multi-com- numerous extrachromosomal nucleoli in the nucleus of ponent molecular complexes such as the ribosome [10]. large in Xenopus laevis oocytes, the dynamics of coales- This challenges the well-worn paradigm of a small num- cence were shown to be quantitatively consistent with ber of molecules undergoing structurally-defined inter- coalescence dynamics of simple liquids, such as two oil actions along ordered assembly pathways. Progress on this droplets fusing in water [15]. Interestingly, this study problem will require expanding the frontiers of how we revealed ATP-dependent dynamics, suggesting a possi- think about intracellular organization. ble form of ‘active diffusion’ within the nucleolus itself. Work in Drosophila oocytes has revealed that novel nu- Assembling nuclear bodies through phase clear bodies can be induced to nucleate and grow from the transitions nucleoplasm by mechanical perturbation of the egg cham- A hypothesis that has gained increasing attention is that ber, which likely changes the nucleoplasmic salt concen- nuclear and cytoplasmic bodies assemble through intra- tration and thus the strength of molecular interactions cellular phase separation [11–14]. Phase transitions are [16]. These nuclear bodies are highly spherical and can be common in nature and familiar from everyday experi- observed to undergo striking liquid-like coalescence ences of dewdrops condensing on blades of grass or water events (Figure 2). freezing into ice. Phase transitions reflect the thermody- namic forces driving a system to its equilibrium configu- A fascinating recent study by Nott et al. provides further ration, manifesting in switch-like transitions of large-scale quantitative support for the role of phase separation in the molecular organization. Such behavior is actually well nucleus [17 ]. This study utilizes a Ddx4 construct, which known in biology, from in vitro protein crystallization, when expressed in vitro assembles into liquid phase dro- where soluble proteins are observed to condense into plets. The assembly of these droplets depends on protein concentrated liquid phases or crystalline solid phases. concentration, salt concentration, and temperature, in a In the cell, proteins could thus similarly transition from manner that can be quantitatively modeled as a phase a dispersed nucleoplasmic state into a localized body, and transition. When YFP::Ddx4 constructs are expressed in Current Opinion in Cell Biology 2015, 34:23–30 www.sciencedirect.com Nuclear body phase transitions Zhu and Brangwynne 25 Figure 2 0s 57s 258s 278s 298s Current Opinion in Cell Biology Nucleation, coarsening and coalescence of induced nuclear bodies. A novel type of nuclear body first described in Ref. [16] is induced to nucleate and grow upon gentle mechanical pressure applied to the nucleus of drosophila oocytes (t = 0 s). Large liquid-like nuclear bodies grow and coalesce upon contact with one another (courtesy Brangwynne, unpublished). The nucleus is approximately 40 mm in diameter. HeLa cells, the protein is transported into the