Live-Cell Imaging and Analysis of Nuclear Body Mobility.

The cell nucleus contains different domains and nuclear bodies, whose position relative to each other inside the nucleus can vary depending on the physiological state of the cell. Changes in the three-dimensional organization are associated with the mobility of individual components of the nucleus. In this chapter, we present a protocol for live-cell imaging and analysis of nuclear body mobility. Unlike other similar protocols, our image analysis pipeline includes non-rigid compensation for global motion of the nucleus before particle tracking and trajectory analysis, leading to precise detection of intranuclear movements. The protocol described can be easily adapted to work with most cell lines and nuclear bodies.

New insights into intranuclear inclusions in thyroid carcinoma: Association with autophagy and with BRAFV600E mutation.

BACKGROUND: Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS: NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS: In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS: NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Crowding-induced formation and structural alteration of nuclear compartments: insights from computer simulations.

Our understanding of the structural and dynamical characteristics of nuclear structures such as chromosomes and nuclear bodies (NBs) has increased significantly in recent days owing to advances in biophysical and biochemical techniques. These techniques include the use of computer simulations, which have provided further physical insights complementary to findings from experiments. In this chapter, we review recent computer simulation studies on the structural alteration of chromosome subcompartments and the formation and maintenance of NBs in the highly crowded cell nucleus. It is found that because of macromolecular crowding, the degree of chromosome compaction changes significantly and the formation of NBs is facilitated. We further discuss the physical consequences of these phenomena, which may be of critical importance in understanding genome processes.

Role of promyelocytic leukemia protein in host antiviral defense.

Several pathways have been implicated in the establishment of antiviral state in response to interferon (IFN), one of which implicates the promyelocytic leukemia (PML) protein. The PML gene has been discovered 20 years ago and has led to new insights into oncogenesis, apoptosis, cell senescence, and antiviral defense. PML is induced by IFN, leading to a marked increase of expression of PML isoforms and the number of PML nuclear bodies (NBs). PML is the organizer of the NBs that contains at least 2 permanent NB-associated proteins, the IFN-stimulated gene product Speckled protein of 100 kDa (Sp100) and death-associated dead protein (Daxx), as well as numerous other transient proteins recruited in these structures in response to different stimuli. Accumulating reports have implicated PML in host antiviral defense and revealed various strategies developed by viruses to disrupt PML NBs. This review will focus on the regulation of PML and the implication of PML NBs in conferring resistance to DNA and RNA viruses. The role of PML in mediating an IFN-induced antiviral state will also be discussed.

Orphan nuclear bodies.

Orphan nuclear bodies are defined as nonchromatin nuclear compartments that have been less well studied compared with other well-characterized structures in the nucleus. Nuclear bodies have traditionally been thought of as uniform distinct entities depending on the protein "markers" they contain. However, it is becoming increasingly apparent that nuclear bodies enriched in different sets of transcriptional regulators share a link to the ubiquitin-proteasome and SUMO-conjugation pathways. An emerging concept is that some orphan nuclear bodies might act as sites of protein modification by SUMO and/or proteasomal degradation of ubiquitin-tagged proteins. By defining a specialized environment for protein modification and degradation, orphan nuclear bodies may increase the capacity of cells to survive under varying environmental conditions.

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response.

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.

Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.

The promyelocytic leukaemia (PML) tumour suppressor protein epitomizes the PML-nuclear body (PML-NB) and is crucially required for the proper assembly of this macromolecular nuclear structure. Unlike other, more specialized subnuclear structures such as Cajal and Polycomb group bodies, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions. PML-NBs are dynamic structures that favour the sequestration and release of proteins, mediate their post-translational modifications and promote specific nuclear events in response to various cellular stresses. Recent data suggest that PML-NBs may be heterogeneous in composition, mobility and function.

Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear bodies in G1.

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100. We demonstrate that MAPPs are also devoid of Daxx and these biochemical changes occur prior to chromatin condensation and coincide with the loss of nuclear membrane integrity. MAPPs are highly mobile, yet do not readily exchange PML protein as demonstrated by fluorescence recovery after photo-bleaching (FRAP). A subset of MAPPs remains associated with mitotic chromosomes, providing a possible nucleation site for PML nuclear body formation in G1. As the nuclear envelope reforms in late anaphase, these nascent PML nuclear bodies accumulate components sequentially, for example Sp100 and SUMO-1 before Daxx. After cytokinesis, MAPPs remain in the cytoplasm long after the reincorporation of splicing components and their disappearance coincides with new PML nuclear body formation even in the absence of new protein synthesis. The PML protein within MAPPs is not degraded during mitosis but is recycled to contribute to the formation of new PML nuclear bodies in daughter nuclei. The recycling of PML protein from one cell cycle to the next via mitotic accumulations may represent a common mechanism for the partitioning of other nuclear bodies during mitosis.

Proteasome inhibitors suppress formation of polyglutamine-induced nuclear inclusions in cultured postmitotic neurons.

At least nine neurodegenerative disorders are caused by expansion of polyglutamine repeats in various genes. This expansion induces the formation of nuclear inclusions (NI) within various cell types. In this study, we developed a model for polyglutamine diseases using primary cultures of sympathetic neurons from the superior cervical ganglia of prenatal rat pups. Transfection with a plasmid encoding 127 glutamine repeats causes NI to develop in approximately 70% of the sympathetic neurons within 6 days. In addition, it causes somatic atrophy and inhibits dendritic growth. The NIs contain ubiquitinated proteins and sequester the molecular chaperone heat shock protein 70 (Hsp70). We found that two specific proteasome inhibitors, lactacystin and CEP1612, suppress thezformation of polyglutamine-induced NI. In addition, lactacystin treatment induced the removal of preexisting NI. Western blotting and immunocytochemistry revealed that lactacystin and CEP1612 strongly induce the expression of Hsp70, whereas less specific proteasome inhibitor such as N-acetyl-Leu-Leu-Norleucinal does not. Coexpression of 127 glutamines with a plasmid encoding wild-type Hsp70 gene resulted in a marked reduction of the percentage of neurons containing NI. In addition, transfection with plasmids encoding mutant Hsp70 blocked the effects of lactacystin. These findings further implicate Hsp70 as a neuroprotective molecule and they suggest the potential utility of certain proteasome inhibitors in the treatment of polyglutamine diseases.

PML nuclear bodies: dynamic sensors of DNA damage and cellular stress.

Promyelocytic leukaemia nuclear bodies (PML NBs) are generally present in all mammalian cells, and their integrity correlates with normal differentiation of promyelocytes. Mice that lack PML NBs have impaired immune function, exhibit chromosome instability and are sensitive to carcinogens. Although their direct role in nuclear activity is unclear, PML NBs are implicated in the regulation of transcription, apoptosis, tumour suppression and the anti-viral response. An emerging view is that they represent sites where multi-subunit complexes form and where post-translational modification of regulatory factors, such as p53, occurs in response to cellular stress. Following DNA damage, several repair factors transit through PML NBs in a temporally regulated manner implicating these bodies in DNA repair. We propose that PML NBs are dynamic sensors of cellular stress, which rapidly disassemble following DNA damage into large supramolecular complexes, dispersing associated repair factors to sites of damage. The dramatically increased total surface area available would enhance interactions between PML-associated factors regulating DNA repair and apoptosis.

Stress responses of PML nuclear domains are ablated by ataxin-1 and other nucleoprotein inclusions.

The polyglutamine diseases are characterized by expansion of triplet CAG repeats that encode polyglutamine tracts in otherwise unrelated proteins. One plausible explanation for the neurodegeneration of these disorders proposes that inclusions of such proteins sequester other significant nuclear proteins in inactive form. The present study shows that PML protein is sequestered by inclusions of the pathogenic mutant form of the polyglutamine protein ataxin-1 and that this sequestration removes from the nucleus the free 0.2-1 microm diameter PML nuclear domains (PML-NDs), together with at least one of their many cargo proteins (Sp100). The present study demonstrates that this sequestration can be effected equally by another nuclear protein, RED, which lacks a polyglutamine tract, but expresses a polar zipper repeat. The sequestered PML-NDs no longer respond to stress signals (heat shock or ionizing radiation) to which they are normally sensitive. In both cases, there is independent evidence that the cells initiate other responses to their injury (nuclear translocation of heat shock protein or generation of gamma-H2AX-rich nuclear foci, respectively). The data thus provide strong evidence that multiple species of nuclear inclusion functionally sequester PML-NDs. This mechanism is likely to distort cellular responses to injury of many different types.

Postnatal development of multivesicular nuclear body in the Shiba goat Sertoli cell: an ultrastructural study.

The multivesicular nuclear body (MNB) within the Sertoli cell nucleolus has been observed in the ruminant testis, but not in other mammalian species. Generally, the MNB is composed of vesicles, tubules and ribosome-like structures. This study has been conducted in order to clarify MNB formation during postnatal development. The testes were obtained from immature Shiba goats at 1, 2, 3, 4, and 5 months old, and from adults. They were fixed with 5% glutaraldehyde, postfixed with 1% OsO4, dehydrated in ethanol and embedded in Araldite-M. The serial cross-sections of seminiferous tubules were morphologically and morphometrically observed using light and transmission electron microscopy. In these Shiba goat testes, the MNB contained vesicles and tubules in various sizes, as well as ribosome-like structures. The volume of each Sertoli cell nucleus at each age (1, 2, 3, 4, 5 months old and adult) was about 269.3 microm3, 327.1 microm3, 361.3 microm3, 431.2 microm3, 525.0 microm3, and 760.4 microm3, respectively. The average number of vesicles per Sertoli cell nucleus was 0, 7.4, 11.1, 12.3, 15.5, and 32.7, respectively. At 1 month old, one or more nucleoli with fibrillar components were identified in the Sertoli cell nucleus. No MNB was observed. At 2 months old, a MNB first appeared, though it was underdeveloped and infrequently encountered. At this stage, a MNB, consisting of a small amount of vesicles and ribosomes, was located in the peripheral region of the nucleus. At later stages (3, 4, and 5 months old), MNBs gradually developed, increased in number, moved from the periphery to the central region of the nucleus, and associated with the nucleus to form a well-developed MNB. In the adults, the Sertoli cell nucleus displayed a well-developed and large-sized MNB situated in the central region.