Nuclear bodies: random aggregates of sticky proteins or crucibles of macromolecular assembly?

Abstract

The principles of self-assembly and self-organization are major tenets of molecular and cellular biology. Governed by these principles, the eukaryotic nucleus is composed of numerous subdomains and compartments, collectively described as nuclear bodies. Emerging evidence reveals that associations within and between various nuclear bodies and genomic loci are dynamic and can change in response to cellular signals. This review will discuss recent progress in our understanding of how nuclear body components come together, what happens when they form, and what benefit these subcellular structures may provide to the tissues or organisms in which they are found.

Figure 1. Diversity of Nuclear Bodies

The cartoon in the center of the figure depicts the nucleus of a higher eukaryote. Interphase chromosomes occupy distinct territories (large irregular shapes). The interchromatin space contains numerous subdomains or bodies (colored dots). (A) The nucleoli of a mouse embryonic fibroblast were stained with anti-fibrillarin (red) and counter-stained for DNA using DAPI (blue). Note that fibrillarin localizes primarily to nucleoli (large blobs) but is also found in Cajal bodies (asterisks). (B) Antibodies targeting the U2B″ protein were used to identify the Cajal body (bright dot) in this Arabidopsis nucleus. Note that the nucleolus shows up as a negatively stained region within the nucleoplasmic U2B″ signal. (C) Anti-FLASH antibodies highlight the two histone locus bodies (bright foci) within this Drosophila S2 cell. (D) Mammalian nuclei, as illustrated by this mouse NIH 3T3 cell, typically contain 10–30 PML bodies, stained here with anti-PML. (E) The perinucleolar compartment (PNC) is shown in this human (HeLa) cell hybridized with an oligo-nucleotide probe targeting hY1 RNA (green). The nucleus is counterstained in blue with DAPI. Note that this RNA localizes to the PNC as well as to the cytoplasm. (F) This human U2OS cell was transfected with YFP-tagged Bmi1, a Polycomb group (PcG) protein that is used as a marker for PcG bodies (green). Counterstaining was performed using Hoechst (blue).

Figure 2. Mechanisms of Nuclear Body Formation

(A) Biological systems are thought to be governed by the principle of self-organization (Camazine et al., 2001), which is distinct from the concept of self-assembly (Worrall et al., 2007). Self-assembly involves formation of stable complexes that essentially reach thermodynamic equilibrium (left). In contrast, self-organization operates on steady-state systems—those that are far from equilibrium (right). As outlined by Misteli (2001), in cell biological terms, self-organization can be defined as: “the capacity of a macromolecular complex or organelle to determine its own structure, based on the functional interactions of its components.” Through this mechanism, which requires a continuous exchange of materials, the cell is capable of generating a stable (steady-state) structure from a set of dynamic components. In the cartoon, the steady-state approximation is met because a constant flux of components is maintained. Factors enter the body from the newly synthesized pool and can exit the structure, perhaps in a modified form (sunbursts). Note that the modifications do not necessarily preclude a given component from rebinding to the structure. (B) The assembly of a nuclear body can follow a hierarchically ordered assembly pathway (top), or components can assemble stochastically by a number of individual pathways (bottom). Note that components can enter singly or as large complexes. Although the order of assembly is random in the stochastic model, it is still predicated on molecular interactions. Thus, loss of a given component could lead to failure to incorporate another component or complex.

Figure 3. Components of Cajal and Histone Locus Bodies

The replication-dependent histone genes are typically clustered in metazoan genomes. The histone locus body (HLB) can be viewed as a nuclear subdomain dedicated to the transcription and processing of histone pre-mRNA. In the Venn diagram above, factors known to localize to HLBs in both primary and cancer cell lines are shown. Factors that localize to Cajal bodies (CBs) are also listed. Note that the U7 snRNP, which is essential for processing of histone pre-mRNA 3′ ends, is localized to the CB in human cancer cell lines but to the HLB in primary cells. Although the term HLB was only recently coined (Liu et al., 2006), the structure was probably first identified in 1981, when Gall and coworkers showed that the “sphere organelle” is bound to the histone gene clusters in the newt, Triturus (Gall et al., 1981). These structures were later termed CBs. The presence of extrachromosomal sphere organelles (also termed CBs) in amphibian oocytes and the absence of markers to distinguish between these two types of structures has also been a hindrance. In 1998, Spradling and coworkers (Calvi et al., 1998) showed that an unknown cyclin E-dependent phosphoepitope (MPM-2) localized to a nuclear domain that was subsequently identified as the HLB (White et al., 2007). The first clear demonstration of a structure located at the mammalian histone gene cluster was by Zhao and coworkers, who found that NPAT, a CDK2-cyclin E substrate, colocalized with the mammalian histone genes (Zhao et al., 2000). Subsequently, another HLB protein, called FLASH, was shown to colocalize with NPAT (Barcaroli et al., 2006).

Figure 4. Modeling Macromolecular Assembly

(A) Formation of the U4/U6 di-snRNP requires extensive base-pairing interactions between U4 and U6 snRNAs. The assembly of U4/U6 di-snRNPs is a necessary step that takes place prior to spliceosome formation. Proteins that bind to these snRNAs are not shown in the reaction scheme. (B) Three-dimensional projection of a HeLa cell nucleus showing a single nucleolus and four Cajal bodies (CBs) within its interior (reprinted with permission from Klingauf et al., 2006). Dimensions are in microns. (C) Through the use of simulated random walks within the nuclear space (excluding the nucleolus), Klingauf et al. (2006) showed that the time for productive assembly of U4/U6 di-snRNPs was greatly accelerated by the presence of one or more CBs; optimal assembly rates were achieved when cells contained three to four CBs.