Calreticulin mRNA and protein are localized to protein bodies in storage maize callus cells

Maize callus cells possess numerous protein bodies which develop as sub-compartments of the endoplasmic reticulum. We localized maize calreticulin mRNAs and protein in maize callus cells using in situ hybridization and immunocytochemistry. Calreticulin mRNAs were selectively targeted to the endoplasmic reticulum (ER) subdomains surrounding protein bodies. Profilin mRNAs, used as a positive control for in situ hybridization experiments, showed distinct and rather diffuse localization pattern. Using both, immunofluorescence and immunogold electron microscopy localization techniques, calreticulin was found to be enriched around and within protein bodies in maize callus storage cells. As a positive control for reticuloplasmins, HDEL antibody revealed labelling of protein bodies and of the nuclear envelope. The identity of protein bodies was confirmed by specific binding of an α zein antibody. These data suggest that calreticulin mRNA is targeted towards protein body forming subdomains of the ER, and that calreticulin is localized and enriched in these protein bodies. The possibility that calreticulin plays an important role in zein retention within the ER and/or its assembly and packaging into protein bodies during protein body biogenesis in maize callus is discussed.     

Nuclear structure and intranuclear retention of premature RNAs

Eukaryotic cells are highly compartmentalized, each compartment being surrounded by a lipid bilayer. This membrane-based organization allows cells to use their volumes to encode information. The lack of intranuclear membranes suggested that the nucleus was largely devoid of structural organization. However, recent work has defined numerous specialized nuclear subdomains. Importantly, RNA processing factors do not display random distribution but cluster in defined nuclear bodies. Although these structures are well characterized morphologically, their function in relation to RNA metabolism remains elusive. In this review, we will discuss the putative participation of nuclear substructures in a quality control step of RNA biogenesis, the nuclear retention of premature RNA.     

Nuclear bodies in neurodegenerative disease

Neurodegenerative diseases are characterized by a relentlessly progressive loss of the functional and structural integrity of the central nervous system. In many cases, these diseases arise sporadically and the causes are unknown. The abnormal aggregation of protein within the cytoplasm or the nucleus of brain cells represents a unifying pathological feature of these diseases. There is increasing evidence for nuclear dysfunction in neurodegenerative diseases. How this relates to protein aggregation in the context of "cause and effect" remains to be determined in most cases. Co-ordinated nuclear function is predicated on the activity of distinct nuclear subdomains, or nuclear bodies, each responsible for a specific function. If nuclear dysfunction represents an important etiopathological feature in neurodegenerative disease, then this should be reflected by functional and/or morphological alterations in this nuclear compartmentalization. For most neurodegenerative diseases, evidence for nuclear dysfunction, with attendant consequences for nuclear architecture, is only beginning to emerge. In this review, I will discuss neurodegenerative diseases in the context of nuclear dysfunction and, more specifically, alterations in nuclear bodies. Although research in this field is in its infancy, identifying alterations in the nucleus in neurodegenerative disease has potentially profound implications for elucidating the pathogenesis of these disorders.     

SUMOrganization of the nucleus

In the eukaryotic nucleus, gene expression and maintenance of genome integrity are tightly controlled at multiple levels, from the molecular details to the higher-order structure of the genome. The nucleus contains spatially and functionally distinct compartments in which these fundamental processes are carried out. While the dynamics and functions of some nuclear subdomains, like the nucleolus, have been well studied, other domains, like the PML-nuclear bodies, remain enigmatic. Recent evidence has now implicated the SUMOylation pathway as an important player in subnuclear architecture, particularly in the assembly of PML-nuclear bodies. Related functions include the organization of chromatin loops and maintenance of rDNA repeat stability. Consequently, complete loss of SUMO modification profoundly affects nuclear organization and cell viability.     

Coilin methylation regulates nuclear body formation

Cajal bodies (CBs) are nuclear suborganelles involved in biogenesis of small RNAs. Twin structures, called gems, contain high concentrations of the survival motor neurons (SMN) protein complex. CBs and gems often colocalize, and communication between these subdomains is mediated by coilin, the CB marker. Coilin contains symmetrical dimethylarginines that modulate its affinity for SMN, and, thus, localization of SMN complexes to CBs. Inhibition of methylation or mutation of the coilin RG box dramatically decreases binding of coilin to SMN, resulting in gem formation. Coilin is hypomethylated in cells that display gems, but not in those that primarily contain CBs. Likewise, extracts prepared from cells that display gems are less efficient in methylating coilin and Sm constructs in vitro. These results demonstrate that alterations in protein methylation status can affect nuclear organization.     

Functional dissection of the phosphorylated termini of fission yeast DNA topoisomerase II

Fission Yeast DNA topoisomerase II (165 kD) consists of an enzymatically active 125-kD core, approximately 10-kD NH2-terminal and 30-kD COOH-terminal domains. The question addressed in the present study is what is the role of the topo II termini. Although deletion of either the NH2 or the COOH terminus is viable, deletion of both termini is lethal; the termini share an essential role for viability. We show here that topo II phosphorylation sites are localized in the terminal domains, but dephosphorylated topo II is still active. The topo II terminal sequences are required for nuclear localization; topo II double terminal deletion mutants are deficient for nuclear targeting, whereas wild-type and single deletion mutant topo IIs are transported into the nucleus with different efficiencies. Functional subdomains in the NH2 terminus are further dissected; we identified a 15 amino acid nuclear localization sequence (NLS) which is essential for viability and nuclear localization when the COOH terminus is deleted. This NLS could be substituted with SV-40 large T-antigen NLS. Two other functional subdomains were found; a non-essential acidic stretch which is phosphorylated and apparently enhances the nuclear localization and an essential hydrophilic stretch of unknown function. Motifs similar to these three NH2-terminal subdomains are also found in the COOH terminus. Our results support the possibility that phosphorylation of topo II does not play an essential role in fission yeast.     

Ligand-selective targeting of the glucocorticoid receptor to nuclear subdomains is associated with decreased receptor mobility

The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.     

Immediate early gene X1 (IEX-1) is organized in subnuclear structures and partially co-localizes with promyelocytic leukemia protein in HeLa cells

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.     

Pumping RNA: nuclear bodybuilding along the RNP pipeline

Cajal bodies (CBs) are nuclear subdomains involved in the biogenesis of several classes of small ribonucleoproteins (RNPs). A number of recent advances highlight progress in the understanding of the organization and dynamics of CB components. For example, a class of small Cajal body-specific (sca) RNPs has been discovered. Localization of scaRNPs to CBs was shown to depend on a conserved RNA motif. Intriguingly, this motif is also present in mammalian telomerase RNA and the evidence suggests that assembly of the active form of telomerase RNP occurs in and around CBs during S phase. Important steps in the assembly and modification of spliceosomal RNPs have also been shown to take place in CBs. Additional experiments have revealed the existence of kinetically distinct subclasses of CB components. Finally, the recent identification of novel markers for CBs in both Drosophila and Arabidopsis not only lays to rest questions about the evolutionary conservation of these nuclear suborganelles, but also should enable forward genetic screens for the identification of new components and pathways involved in their assembly, maintenance and function.     

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly

Transmission of malaria-causing parasites to mosquitoes relies on the production of gametocyte stages and their development into gametes. These stages display various microtubule cytoskeletons and the architecture of the corresponding microtubule organisation centres (MTOC) remains elusive. Combining ultrastructure expansion microscopy (U-ExM) with bulk proteome labelling, we first reconstructed in 3D the subpellicular microtubule network which confers cell rigidity to Plasmodium falciparum gametocytes. Upon activation, as the microgametocyte undergoes three rounds of endomitosis, it also assembles axonemes to form eight flagellated microgametes. U-ExM combined with Pan-ExM further revealed the molecular architecture of the bipartite MTOC coordinating mitosis with axoneme formation. This MTOC spans the nuclear membrane linking cytoplasmic basal bodies to intranuclear bodies by proteinaceous filaments. In P. berghei, the eight basal bodies are concomitantly de novo assembled in a SAS6- and SAS4-dependent manner from a deuterosome-like structure, where centrin, γ-tubulin, SAS4 and SAS6 form distinct subdomains. Basal bodies display a fusion of the proximal and central cores where centrin and SAS6 are surrounded by a SAS4-toroid in the lumen of the microtubule wall. Sequential nucleation of axonemes and mitotic spindles is associated with a dynamic movement of γ-tubulin from the basal bodies to the intranuclear bodies. This dynamic architecture relies on two non-canonical regulators, the calcium-dependent protein kinase 4 and the serine/arginine-protein kinase 1. Altogether, these results provide insights into the molecular organisation of a bipartite MTOC that may reflect a functional transition of a basal body to coordinate axoneme assembly with mitosis.     

Further ultrastructural characterization of the intranuclear ring-shaped bodies of the plant Lacandonia schismatica

Ring-shaped bodies are found in the nucleus of Lacandonia schismatica, a rare plant with the sexual organs inverted. They are 0.5-microm-diameter structures that present an electron-dense external ring surrounding a central core. Ultrastructural studies indicate that these bodies contain RNA. The external ring is labeled with antibodies against small nuclear ribonucleoproteins, suggesting that they may be involved in pre-mRNA metabolism. In the present work we further characterized these intranuclear ring-shaped structures by serial-sectioning analysis. Moreover, we tested the presence of additional molecular elements related to pre-mRNA metabolism, such as SR proteins and poly(A)(+) RNA, using immunoelectron microscopy and ultrastructural in situ hybridization. Our results show that these nuclear bodies are spherical. They contain SR proteins involved in splicing and postsplicing events and little to no poly(A)(+) RNA. We also found similar nuclear bodies in other plant and animal species. Therefore, ring-shaped bodies in L. schismatica are spherical, highly compartmentalized nuclear structures that may be involved in pre-mRNA metabolism.