Biomedical subjects

Roeland W Dirks

Publications and source records attributed to Roeland W Dirks.

13 recordsLinked to original sources

PubMed2006

Changes in lamina structure are followed by spatial reorganization of heterochromatic regions in caspase-8-activated human mesenchymal stem cells.

Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.

Blotting, Western↗

PubMed2006

Regulation of sexual development of Plasmodium by translational repression.

Translational repression of messenger RNAs (mRNAs) plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. Translational repression and mRNA turnover were shown to influence stage-specific gene expression in the protozoan Plasmodium. The DDX6-class RNA helicase, DOZI (development of zygote inhibited), is found in a complex with mRNA species in cytoplasmic bodies of female, blood-stage gametocytes. These translationally repressed complexes are normally stored for translation after fertilization. Genetic disruption of pbdozi inhibits the formation of the ribonucleoprotein complexes, and instead, at least 370 transcripts are diverted to a degradation pathway.

Animals↗

PubMed2006

Styryl molecules light-up RNAs.

A combinatorial library of 1336 fluorescent styryl molecules was synthesized aiming to select dyes that are photostable, non-toxic, and specific for RNA molecules in living cells . These dyes are potentially important to the study of gene expression in live cells.

Animals↗

PubMed2006

Advances in fluorescent tracking of nucleic acids in living cells.

Nucleic acids are typically detected in morphologically preserved fixed cells and tissues using in situ hybridization techniques. This review discusses a variety of established and more challenging fluorescence-based methods for the detection and tracking of DNA or RNA sequences in living cells. Over the past few years, various fluorescent in vivo labeling methods have been developed, and dedicated microscope and image analysis tools have been designed. These advances in technologies indicate that live-cell imaging of nucleic acids is likely to become a standard research tool for understanding genome organization and gene expression regulation in the near future. Recent live-cell imaging studies have already provided important insights into the dynamic behaviors of chromatin and RNAs in the cell.

Biological Transport↗

PubMed2005

A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain.

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

Animals↗

PubMed2005

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.

Animals↗

PubMed2005

FISH and immunocytochemistry: towards visualising single target molecules in living cells.

Knowledge of how molecules interact in space and time is crucial for understanding cellular processes. A host of novel techniques have been developed for the visualisation of single target molecules in living cells, many based on fluorescence in situ hybridisation (FISH) or immunocytochemistry (IC). To extend the applicability of FISH to living cells, special backbone-modified probes and specific conformations (molecular beacons) have been designed. In the case of IC, conventional immunoreagents have been fine-tuned with respect to size and affinity or replaced with new protein scaffolds based on ankyrin repeat proteins. Other key advances include the use of proximity ligation to confirm vicinity binding and the use of quantum dots, which have proven potential for cellular labelling.

Cell Physiological Phenomena↗

PubMed2004

Poly(A)+ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells.

Many of the protein factors that play a role in nuclear export of mRNAs have been identified, but still little is known about how mRNAs are transported through the cell nucleus and which nuclear compartments are involved in mRNA transport. Using fluorescent 2'O-methyl oligoribonucleotide probes, we investigated the mobility of poly(A)+ RNA in the nucleoplasm and in nuclear speckles of U2OS cells. Quantitative analysis of diffusion using photobleaching techniques revealed that the majority of poly(A)+ RNA move throughout the nucleus, including in and out of speckles (also called SC-35 domains), which are enriched for splicing factors. Interestingly, in the presence of the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, the association of poly(A)+ RNA with speckles remained dynamic. Our results show that RNA movement is energy dependent and that the proportion of nuclear poly(A)+ RNA that resides in speckles is a dynamic population that transiently interacts with speckles independent of the transcriptional status of the cell. Rather than the poly(A)+ RNA within speckles serving a stable structural role, our findings support the suggestion of a more active role of these regions in nuclear RNA metabolism and/or transport.

Active Transport, Cell Nucleus↗

PubMed2003

Visualizing telomere dynamics in living mammalian cells using PNA probes.

Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A fluorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to specifically associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital fluorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed significant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.

Base Sequence↗

PubMed2003

Visualizing RNA molecules inside the nucleus of living cells.

Fluorescence in situ hybridization is a widely used technique in cell biology providing insight into the spatial organization of specific RNA transcripts in the cell nucleus. However, to further investigate the dynamics of the transcription process and the transport rates of RNAs through the nucleus, RNAs need to be visualized and tracked in the living cell. In past years, various methods have been developed with the aim of tagging specific RNAs with a fluorescent moiety without interfering with cell vitality. These methods include the delivery of probes into a living cell, the in vivo hybridization of fluorescent oligonucleotide probes to endogenous RNAs, and the microscopic imaging of the tagged RNAs in living cells. In this article, we review a number of methods for tagging and visualizing endogenous RNAs in living cells. In addition, a protocol is described that allows detection of various RNA types using fluorochrome-labeled 2(')-O-methyl oligoribonucleotide (2(')-OMe RNA) probes. Compared with conventional oligodeoxynucleotide probes, 2(')-OMe RNA probes are not degraded by nucleases, form stable hybrids with structured RNAs, and do not interfere with cell vitality.

Animals↗

PubMed2002

Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts.

Compelling evidence supports an intimate link in time and space between eukaryotic pre-mRNA synthesis and processing and nucleocytoplasmic transport of mature mRNA. In this study, we analyzed the kinetic behavior of these processes in a quantitative manner. We used FISH and confocal scanning laser microscopy to detect transcripts produced by an inducible human cytomegalovirus immediate early (HCMV-IE) expression system. Upon induction, a large amount of pre-mRNA accumulated in nuclear foci at or near their transcription sites and, at later time, throughout the nucleoplasm. Inhibition of RNA polymerase II activity resulted in a rapid decrease in the number of transcripts in the nuclear RNA foci (half time approximately two minutes), indicating that accumulated transcripts were rapidly spliced and then released. The dispersed nucleoplasmic transcripts exited the nucleus with a half time of approximately 10 minutes. Both processes were temperature dependent, suggesting that mRNA export is an active process. RNA polymerase II activation revealed that production of mature HCMV IE mRNAs required less than five minutes. Transcripts radiated from the gene at an average speed of approximately 0.13 microm(2)/sec from this time on. Thus, it appears that these processes are tightly linked in time and space, with the splicing reaction as a rate-limiting factor.

Active Transport, Cell Nucleus↗

PubMed2002

Induction of p21 mRNA synthesis after short-wavelength UV light visualized in individual cells by RNA FISH.

Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation. (J Histochem Cytochem 50:81-89, 2002)

Cell Line↗

PubMedDate not supplied

Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not.

PML bodies are nuclear organelles that are associated with various diseases and are suggested to be involved in multiple cellular activities including transcriptional regulation, apoptosis, and antiviral defence. Because many proteins with different functions aggregate in PML bodies, it has also been suggested that these bodies function as nuclear depots. Some proteins consistently found in PML bodies may form a stable scaffold that regulates the recruitment of other proteins. Thus, some proteins might be stably integrated into PML bodies while others continuously exchange with the nucleoplasm. To study the dynamic properties of PML bodies and resident proteins, we constructed fusion proteins of Sp100, PML, and CBP with autofluorescent proteins. Using time-lapse imaging, we show that PML bodies exhibit little movement but that small foci that contain Sp100 but not PML are dynamic and fuse with PML bodies. Furthermore, we show by monitoring fluorescence recovery after photobleaching that Sp100, PML, and CBP are dynamic components of PML bodies. This suggests that these proteins do not play a strict structural role in these bodies but that they function at other sites in the nucleoplasm.

Antigens, Nuclear↗