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PubMed2005

Subnuclear organelles: new insights into form and function.

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.

Animals↗
PubMed2004

snRNPs are present in the karyosome capsule in the weevil germinal vesicle.

Within the oocyte nucleus of the apple blossom weevil, Anthonomus pomorum (Insecta, Coleoptera) highly condensed and transcriptionaly inactive chromosomes form the karyosome. During its formation, within the nucleoplasm numerous, variably sized spherical inclusions termed nuclear bodies occur. As oogenesis progresses, the karyosome is gradually surrounded by a prominent sheath, the karyosome capsule. The function and molecular composition of both the nuclear bodies and the karyosome capsule are largely unknown. Using cytochemical methods we demonstrate that DNA is confined to the karyosome and there is no extrachromosomal DNA accumulations within the nucleoplasm. In addition, none of the oocyte nucleus subdomains contain argyrophilic proteins. Our immunoEM study revealed that in contrast to similar structures in germinal vesicles in other insect species, the nuclear bodies of A. pomorum do not cross-react with antibodies recognising small nuclear ribonucleoproteins, coilin or the splicing factor SC-35. Unexpectedly, we found that as the karyosome capsule develops, mature small nuclear RNAs and proteins containing the Sm epitope associate with the capsule material. We suggest that the karyosome capsule is a storage site for small nuclear ribonucleoprotein particles, which may be used during early embryonic development.

Animals↗
PubMed2005

Nuclear envelope remodeling during mouse spermiogenesis: postmeiotic expression and redistribution of germline lamin B3.

Lamins are members of a multigene family of structural nuclear envelope (NE) proteins. Differentiated mammalian somatic cells express lamins A, C, B1, and B2. The composition and organization of the nuclear lamina of mammalian spermatogenic cells differ significantly from that of somatic cells as they express lamin B1 as well as two short germ line-specific isoforms, namely lamins B3 and C2. Here we describe in detail the expression pattern and localization of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, we show that lamin B3 is selectively expressed during spermiogenesis (i.e., postmeiotic stages of spermatogenesis). In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. Our results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely the ubiquitous lamin B1 and the germline-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis.

Age Factors↗
PubMed2006

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.

Animals↗
PubMed2006

Recruitment of phosphorylated small heat shock protein Hsp27 to nuclear speckles without stress.

During stress, the mammalian small heat shock protein Hsp27 enters cell nuclei. The present study examines the requirements for entry of Hsp27 into nuclei of normal rat kidney (NRK) renal epithelial cells, and for its interactions with specific nuclear structures. We find that phosphorylation of Hsp27 is necessary for the efficient entry into nuclei during heat shock but not sufficient for efficient nuclear entry under control conditions. We further report that Hsp27 is recruited to an RNAse sensitive fraction of SC35 positive nuclear speckles, but not other intranuclear structures, in response to heat shock. Intriguingly, Hsp27 phosphorylation, in the absence of stress, is sufficient for recruitment to speckles found in post-anaphase stage mitotic cells. Additionally, pseudophosphorylated Hsp27 fused to a nuclear localization peptide (NLS) is recruited to nuclear speckles in unstressed interphase cells, but wildtype and nonphosphorylatable Hsp27 NLS fusion proteins are not. The expression of NLS-Hsp27 mutants does not enhance colony forming abilities of cells subjected to severe heat shock, but does regulate nuclear speckle morphology. These data demonstrate that phosphorylation, but not stress, mediates Hsp27 recruitment to an RNAse soluble fraction of nuclear speckles and support a site-specific role for Hsp27 within the nucleus.

Active Transport, Cell Nucleus↗
PubMed2004

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein.

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10AC1. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10AC1 gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of approximately 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10AC1, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10AC1 proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10AC1 protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10AC1 gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site.

Alleles↗
PubMed2003

New type of snRNP containing nuclear bodies in plant cells.

In larch (Larix decidua Mill.) microspores a new type of nuclear bodies has been found which are an element of the spatial organization of the splicing system in plant cell. These are bizonal bodies, ultrastructurally differentiated into a coiled part and a dense part. Using immunocytochemistry and in situ hybridization at the EM level, the coiled part of the bizonal body was found to contain snRNA including U2 snRNA, Sm proteins and nucleolar proteins of the agyrophilic type and fibrillarin. The dense part contains Sm proteins but lacks snRNA. Such a separation of macromolecules related to splicing occurring within the bizonal bodies microspore is striking by the similarity of these bodies to amphibian oocyte snurposomes. The occurrence in plant cells, beside widely known coiled bodies (CBs), also of other nuclear bodies related to splicing proves that in plants similarly as for animals the differentiation among domains containing elements of the splicing system occurs.

Cell Nucleolus↗
PubMed2003

Intranuclear Ca2+ signals within individual nuclear lobes of neutrophils.

Cytosolic free Ca(2+)and intranuclear Ca(2+)behave similarly in human neutrophils. However, conventional laser scanning at 350 ms/frame resolution at lower than physiological temperatures demonstrates that (i) the nuclear fluo3-Ca(2+)signal persists longer than the cytosolic signal in some (but not all) nuclear lobes, (ii) the neutrophil nuclear membrane and fine inter-lobe filaments present barriers to diffusion of fluo3-Ca(2+)and lucifer yellow, and (iii) the diffusion barrier correlates with condensed chromosomal material on the nuclear envelope and blockage of the movement of fluo3-Ca(2+)into individual nuclear lobes.

Calcium Signaling↗
PubMed2005

FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles.

Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.

Active Transport, Cell Nucleus↗
PubMed2005

Dynamic positioning of the fission yeast cell division plane.

A key question in cytokinesis is how the cell division plane is positioned. Whereas microtubules of the mitotic apparatus specify the division site in animal cells, we show here that the nucleus plays this role in the fission yeast Schizosaccharomyces pombe. By centrifuging cells to move the nucleus, we find that the nucleus (or a nuclear-associated structure) actively influences the position of contractile ring assembly during early mitosis. Displacement of the nucleus during this induction period can lead to formation of multiple rings. The nucleus signals its position in a microtubule-independent manner by emitting the protein mid1p. Furthermore, movement of ring fragments together minimizes formation of multiple division sites. These dynamic mechanisms of ring positioning provide a robust coordination of nuclear and cell division.

Cell Nucleus↗
PubMed2005

p300/CBP-associated factor drives DEK into interchromatin granule clusters.

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Acetylation↗
PubMed2006

Deficiency of the zinc finger protein ZPR1 causes defects in transcription and cell cycle progression.

The zinc finger protein ZPR1 is present in both the cytoplasm and nucleoplasm. Cell cycle analysis demonstrates that ZPR1 undergoes major changes in subcellular distribution during proliferation. ZPR1 is diffusely localized throughout the cell during the G(1) and G(2)/M phases of the cell cycle. In contrast, ZPR1 redistributes to the nucleus during S phase and ZPR1 exhibits prominent co-localization with the survival motor neurons protein and the histone gene-specific transcription factor NPAT in subnuclear foci, including Cajal bodies that associate with histone gene clusters. ZPR1 deficiency causes disruption of survival motor neurons and NPAT localization within the nucleus, blocks S phase progression, and arrests cells in both the G(1) and G(2) phases of the cell cycle. These changes in subnuclear architecture and cell cycle progression may be caused by transcriptional defects in ZPR1-deficient cells, including decreased histone gene expression.

Carrier Proteins↗
PubMedDate not supplied

Ultrastructural preservation of ovarian cortical tissue cryopreserved in dimethylsulfoxide for subsequent transplantation into young female cancer patients.

Cytotoxic therapy in young women with cancer may cause loss of ovarian function, and ovarian cryopreservation has been proposed as a means of preserving fertility. The objective of this study is to assess ultrastructural preservation of follicles in frozen-thawed ovarian cortical strips, previously cryopreserved using dimethylsulfoxide and a standard slow-cool/rapid-thaw protocol. Ovarian cortical strips (patients, n=7) were thawed and fixed in glutaraldehyde for epoxy resin embedding electron microscopy according to conventional procedures. Oocytes were generally well preserved, on the basis of plasma-membrane integrity, uniformity of chromatin pattern, mitochondrial and rough endoplasmic reticulum (rER) cisternal integrity, and absence of cytoplasmic and intranuclear clear spaces. Frequently, rER cisternae were distended and intramitochondrial matrices lost. Granulosa cells showed somewhat variable preservation. Some were dark-staining; others exhibited washed-out cytoplasm containing damaged or artifactually expanded organelles. The consistently good preservation of oocytes and the good but more variable preservation of granulosa cells provides scientific validation of one component in the overall clinical procedure of attempting to reestablish fertility in young female patients after cytotoxic therapy for cancer.

Adult↗
PubMed2005

A multistep, GTP-driven mechanism controlling the dynamic cycling of nucleostemin.

Nucleostemin (NS) was identified as a stem cell- and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra- and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner.

Amino Acid Sequence↗
PubMed2005

Going in GTP cycles in the nucleolus.

Proteins are directed to cellular compartments by specific localization signals. A GTP-driven cycle has now been identified as a mechanism for protein targeting to the nucleolus. The involvement of a GTP switch suggests that nucleolar localization can be regulated and may be responsive to extracellular stimuli via signaling pathways. The uncovered mechanism also implies that localization is determined by increased retention rather than directed targeting.

Carrier Proteins↗
PubMed2006

In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains.

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.

Active Transport, Cell Nucleus↗
PubMed2007

Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts.

In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 alpha (LAP2alpha) upon entry and exit from G(0) is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2alpha are down-regulated in G(0). Although RbS780 phosphoform and LAP2alpha are up-regulated upon reentry into G(1) and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2alpha is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G(1) phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2alpha or lamin A/C in HDFs leads to accumulation of Rb in speckles and G(1) arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2alpha and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.

Cell Cycle↗