http://repub.eur.nl/res/aut/2019/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/31143/ 2011-08-22T00:00:00Z RAD18 is an ubiquitin ligase involved in replicative damage bypass and DNA double-strand break (DSB) repair processes. We found that RPA is required for the dynamic pattern of RAD18 localization during the cell cycle, and for accumulation of RAD18 at sites of γ-irradiation-induced DNA damage. In addition, RAD18 colocalizes with chromatin-associated conjugated ubiquitin and ubiquitylated H2A throughout the cell cycle and following irradiation. This localization pattern depends on the presence of an intact, ubiquitin-binding Zinc finger domain. Using a biochemical approach, we show that RAD18 directly binds to ubiquitylated H2A and several other unknown ubiquitylated chromatin components. This interaction also depends on the RAD18 Zinc finger, and increases upon the induction of DSBs by γ-irradiation. Intriguingly, RAD18 does not always colocalize with regions that show enhanced H2A ubiquitylation. In human female primary fibroblasts, where one of the two X chromosomes is inactivated to equalize X-chromosomal gene expression between male (XY) and female (XX) cells, this inactive X is enriched for ubiquitylated H2A, but only rarely accumulates RAD18. This indicates that the binding of RAD18 to ubiquitylated H2A is context-dependent. Regarding the functional relevance of RAD18 localization at DSBs, we found that RAD18 is required for recruitment of RAD9, one of the components of the 9-1-1 checkpoint complex, to these sites. Recruitment of RAD9 requires the functions of the RING and Zinc finger domains of RAD18. Together, our data indicate that association of RAD18 with DSBs through ubiquitylated H2A and other ubiquitylated chromatin components allows recruitment of RAD9, which may function directly in DSB repair, independent of downstream activation of the checkpoint kinases CHK1 and CHK2. http://repub.eur.nl/res/pub/31231/ 2011-08-15T00:00:00Z RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs. http://repub.eur.nl/res/pub/34467/ 2011-08-15T00:00:00Z Aims: The study of the intracellular oxido-reductive (redox) state is of extreme relevance to the dopamine (DA) neurons of the substantia nigra pars compacta. These cells possess a distinct physiology intrinsically associated with elevated reactive oxygen species production, and they selectively degenerate in Parkinson's disease under oxidative stress conditions. To test the hypothesis that these cells display a unique redox response to mild, physiologically relevant oxidative insults when compared with other neuronal populations, we sought to develop a novel method for quantitatively assessing mild variations in intracellular redox state. Results: We have developed a new imaging strategy to study redox variations in single cells, which is sensitive enough to detect changes within the physiological range. We studied DA neurons' physiological redox response in biological systems of increasing complexity-from primary cultures to zebrafish larvae, to mammalian brains-and identified a redox response that is distinctive for substantia nigra pars compacta DA neurons. We studied simultaneously, and in the same cells, redox state and signaling activation and found that these phenomena are synchronized. Innovation: The redox histochemistry method we have developed allows for sensitive quantification of intracellular redox state in situ. As this method is compatible with traditional immunohistochemical techniques, it can be applied to diverse settings to investigate, in theory, any cell type of interest. Conclusion: Although the technique we have developed is of general interest, these findings provide insights into the biology of DA neurons in health and disease and may have implications for therapeutic intervention. http://repub.eur.nl/res/pub/33698/ 2011-04-01T00:00:00Z Background & Aims:99mTc-mebrofenin hepatobiliary scintigraphy (HBS) and the indocyanine green (ICG) clearance test are used for the assessment of hepatic function before and after liver surgery. The hepatic uptake of99mTc-mebrofenin and ICG is considered similar to the uptake of organic anions such as bilirubin and bile acids. Little is known about hepatic uptake mechanisms of both compounds and recent evidence suggests that the hepatic transporters for ICG and99mTc-mebrofenin are distinct. The aim of this study was to identify the specific human hepatic transporters of99mTc-mebrofenin and ICG. Methods: The uptake of99mTc-mebrofenin was investigated in cRNA-injected Xenopus laevis oocytes expressing human OATP1B1, OATP1B3, OATP2B1, or NTCP. Chinese hamster ovary (CHO) cells stably expressing OATP1B1, OATP1B3, OATP2B1, or NTCP were used as a mammalian expression system. ICG transport into CHO cells was additionally imaged with confocal microscopy. Results: We demonstrated that OATP1B1 and OATP1B3 are involved in the transport of99mTc-mebrofenin. OATP1B1 showed an approximately 1.5-fold higher affinity for99mTc-mebrofenin compared to OATP1B3. ICG is transported by OATP1B3 and NTCP. Conclusions: The transporter specificity of99mTc-mebrofenin and ICG partially overlaps as both compounds are transported by OATP1B3.99mTc- mebrofenin is also taken up by OATP1B1, whereas ICG is additionally transported by NTCP. http://repub.eur.nl/res/pub/33502/ 2011-03-07T00:00:00Z Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus. http://repub.eur.nl/res/pub/32826/ 2010-09-01T00:00:00Z Background-We imaged the protease activity of matrix metalloproteinases (MMPs) upregulated during aneurysm formation, using protease-activatable near-infrared fluorescence probes. We tested whether these protease-activatable sensors can directly report the in vivo activity of the key biomarkers in aneurysm, using our genetically modified fibulin-4 mouse models for aneurysm formation. Mice homozygous for the fibulin-4 reduced-expression allele (fibulin-4R/R) show dilatation of the ascending aorta and a tortuous, stiffened aorta resulting from disorganized elastic fiber networks. Strikingly, even a moderate reduction in expression of fibulin-4 in the heterozygous fibulin-4+/Rmice occasionally results in modest aneurysm formation. Methods and Results-Aorta transcriptome and protein expression analysis of fibulin-4+/Rand fibulin-4R/Ranimals identified excessive transforming growth factor-β signaling as the critical event in the pathogenesis of aneurysm formation. To determine whether a perturbed elastin lamellar structure arose from induction of transforming growth factor-β-regulated MMPs, we performed gelatin zymography and used a protease-activatable near-infrared fluorescence probe to monitor and quantify MMP upregulation in animals, using various in vivo optical imaging modules and coregistration of the fluorescence signal with CT images of the same animals. Gelatin zymography demonstrated a significant increase in the presence of the active form of MMP-9 in the aortic arch of fibulin-4R/Rmice. In vivo analysis of MMP upregulation using the near-infrared fluorescence probe and subsequent isosurface concentration mapping from reconstructed tomographic images from fibulin-4+/Rand fibulin-4R/Rmice revealed a graded increase in activation of MMPs within the aneurysmal lesions. Conclusions-We aimed to develop molecular imaging procedures for faster, earlier, and easier recognition of aortic aneurysms. We show that in vivo coregistration of MMP activity by noninvasive tomographic imaging methods allows the detection of increased MMP activity, even before the aneurysm has actually formed. http://repub.eur.nl/res/pub/28411/ 2010-08-11T00:00:00Z Motivation: Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Results: Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. http://repub.eur.nl/res/pub/27321/ 2010-02-16T00:00:00Z Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1+, c-kit+, CD41+) directly from ventral aortic haemogenic endothelial cells. http://repub.eur.nl/res/pub/19524/ 2010-02-01T00:00:00Z The cytoplasmic chromatoid body (CB) organizes mRNA metabolism and small regulatory RNA pathways, in relation to haploid gene expression, in mammalian round spermatids. However, little is known about functions and fate of the CB at later steps of spermatogenesis, when elongating spermatids undergo chromatin compaction and transcriptional silencing. In mouse elongating spermatids, we detected accumulation of the testis-specific serine/threonine kinases TSSK1 and TSSK2, and the substrate TSKS, in a ring-shaped structure around the base of the flagellum and in a cytoplasmic satellite, both corresponding to structures described to originate from the CB. At later steps of spermatid differentiation, the ring is found at the caudal end of the newly formed mitochondrial sheath. Targeted deletion of the tandemly arranged genes Tssk1 and Tssk2 in mouse resulted in male infertility, with loss of the CB-derived ring structure, and with elongating spermatids possessing a collapsed mitochondrial sheath. These results reveal TSSK1- and TSSK2-dependent functions of a transformed CB in post-meiotic cytodifferentiation of spermatids. http://repub.eur.nl/res/pub/17041/ 2009-10-01T00:00:00Z The past decade has seen an unprecedented data explosion in biology. It has become evident that in order to take full advantage of the potential wealth of information hidden in the data produced by even a single experiment, visual inspection and manual analysis are no longer adequate. To ensure efficiency, consistency, and completeness in data processing and analysis, computational tools are essential. Of particular importance to many modern live-cell imaging experiments is the ability to automatically track and analyze the motion of objects in time-lapse microscopy images. This article surveys the recent literature in this area. Covering all scales of microscopic observation, from cells, down to molecules, and up to entire organisms, it discusses the latest trends and successes in the development and application of computerized tracking methods in cell and developmental biology. http://repub.eur.nl/res/pub/24318/ 2009-02-01T00:00:00Z The ubiquitin ligase RAD18 is involved in different DNA repair processes. Here, we show that in G1 phase, human RAD18 accumulates in a few relatively large spontaneous foci that contain proteins involved in double-strand break (DSB) repair. These foci persist until cells enter S phase, when numerous small foci appear. At these sites, only 20% of RAD18 colocalizes with PCNA, a known RAD18 substrate. In late G2 phase, RAD18 relocates to nucleoli. After UVC irradiation, PCNA accumulates at the damaged site, followed by RAD18, independent of the cell cycle phase. After induction of DSBs, using low-power multi-photon laser, RAD18 accumulated at the DSB sites, but no PCNA accumulation was observed. Our data show that RAD18 accumulates on DSBs independent of the cell cycle phase. DSBs marked by RAD18 and RAD51 are also positive for RPA in G1 phase, and these DSBs persist until S phase. In addition, we show that DSBs generated in G2 phase are not all repaired, and are observed again in the next G1 phase. We conclude that repair of induced and spontaneous DSBs that accumulate RAD18 and RAD51 in G1 phase cells is delayed until S phase. http://repub.eur.nl/res/pub/29081/ 2008-05-01T00:00:00Z In meiotic prophase of male placental mammals, the heterologous X and Y chromosomes remain largely unsynapsed, which activates meiotic sex chromosome inactivation (MSCI), leading to formation of the transcriptionally silenced XY body. MSCI is most likely related to meiotic silencing of unsynapsed chromatin (MSUC), a mechanism that can silence autosomal unsynapsed chromatin. However, heterologous synapsis and escape from silencing also occur. In mammalian species, formation of DNA double strand breaks (DSBs) during leptotene precedes meiotic chromosome pairing. These DSBs are essential to achieve full synapsis of homologous chromosomes. We generated 25% extra meiotic DSBs by whole body irradiation of mice. This leads to a significant increase in meiotic recombination frequency. In mice carrying translocation chromosomes with synaptic problems, we observed an approximately 35% increase in asynapsis and MSUC of the nonhomologous region in the smallest chromosome pair following irradiation. However, the same nonhomologous region in the largest chromosome pair, shows complete synapsis and escape from MSUC in almost 100% of the nuclei, irrespective of exposure to irradiation. We propose that prevention of synapsis and associated activation of MSUC is linked to the presence of unrepaired meiotic DSBs in the nonhomologous region. Also, spreading of synaptonemal complex formation from regions of homology may act as an opposing force, and drive heterologous synapsis. http://repub.eur.nl/res/pub/17901/ 2008-02-18T00:00:00Z Microtubule (MT) plus end – tracking proteins (+TIPs) specifi cally recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specifi city of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affi nity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fl uorescence-based approaches, we show that two +TIPs, CLIP-170 and endbinding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affi nity. We propose that the observed loss of fl uorescent +TIPs at plus ends does not refl ect the behavior of single molecules but is a result of overall structural changes of the MT end. http://repub.eur.nl/res/pub/35271/ 2007-08-01T00:00:00Z Live cell studies of DNA repair mechanisms are greatly enhanced by new developments in real-time visualization of repair factors in living cells. Combined with recent advances in local sub-nuclear DNA damage induction procedures these methods have yielded detailed information on the dynamics of damage recognition and repair. Here we analyze and discuss the various types of DNA damage induced in cells by three different local damage induction methods: pulsed 800 nm laser irradiation, Hoechst 33342 treatment combined with 405 nm laser irradiation and UV-C (266 nm) laser irradiation. A wide variety of damage was detected with the first two methods, including pyrimidine dimers and single- and double-strand breaks. However, many aspects of the cellular response to presensitization by Hoechst 33342 and subsequent 405 nm irradiation were aberrant from those to every other DNA damaging method described here or in the literature. Whereas, application of low-dose 266 nm laser irradiation induced only UV-specific DNA photolesions allowing the study of the UV-C-induced DNA damage response in a user-defined area in cultured cells. http://repub.eur.nl/res/pub/13946/ 2005-11-01T00:00:00Z The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair. http://repub.eur.nl/res/pub/13948/ 2005-10-15T00:00:00Z CLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP-170 is detected in lungs and embryos of homozygous CLIP-170 knockout mice, but not in other tissues and cell types, indicating that we have generated a hypomorphic mutant. Homozygous CLIP-170 knockout mice are viable and appear normal. However, male knockout mice are subfertile and produce sperm with abnormal heads. Using the knock-in mice, we followed GFP-CLIP-170 expression and behavior in dissected, live testis tubules. We detect plus-end-tracking GFP-CLIP-170 in spermatogonia. As spermatogenesis proceeds, GFP-CLIP-170 expression increases and the fusion protein strongly marks syncytia of differentiated spermatogonia and early prophase spermatocytes. Subsequently GFP-CLIP-170 levels drop, but during spermiogenesis (post-meiotic development), GFP-CLIP-170 accumulates again and is present on spermatid manchettes and centrosomes. Bleaching studies show that, as spermatogenesis progresses, GFP-CLIP-170 converts from a mobile plus-end-tracking protein to a relatively immobile protein. We propose that CLIP-170 has a structural function in the male germline, in particular in spermatid differentiation and sperm head shaping. http://repub.eur.nl/res/pub/13590/ 2005-02-01T00:00:00Z The position of chromosomal neighborhoods in living cells was followed using three different methods for marking chromosomal domains occupying arbitrary locations in the nucleus; photobleaching of GFP-labeled histone H2B, local UV-marked DNA, and photobleaching of fluorescently labeled DNA. All methods revealed that global chromosomal organization can be reestablished through one cell division from mother to daughters. By simultaneously monitoring cell cycle stage in the cells in which relative chromosomal domain positions were tracked, we observed that chromosomal neighborhood organization is apparently lost in the early G1 phase of the cell cycle. However, the daughter cells eventually regain the general chromosomal organization pattern of their mothers, suggesting an active mechanism could be at play to reestablish chromosomal neighborhoods. http://repub.eur.nl/res/pub/13595/ 2004-12-01T00:00:00Z In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin. http://repub.eur.nl/res/pub/13470/ 2004-11-01T00:00:00Z The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake. http://repub.eur.nl/res/pub/3218/ 2004-07-05T00:00:00Z The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active. http://repub.eur.nl/res/pub/13407/ 2004-06-01T00:00:00Z The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals. http://repub.eur.nl/res/pub/10331/ 2004-01-01T00:00:00Z The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively kills Leydig cells is poorly understood. To characterize further the cell-specific actions of EDS, we studied biochemical and morphological changes during apoptosis in different Leydig cell and non-steroidogenic cell models.Rat testicular and H540 tumor Leydig cells were killed by 1-2 mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher concentration of EDS was also necessary for activation of apoptosis in non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney cells were resistant. These variable effects of EDS on apoptosis were independent of new protein synthesis and, interestingly, could be delayed by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also observed chromosomal fragmentation and other hallmarks indicative of apoptosis as evidenced by DNA laddering and fluorescent microscopy. Time-lapse photography with a confocal microscope showed that the time of onset, duration and even the sequence of apoptotic events between individual H540 cells was heterogeneous. When the dose of EDS was gradually increased from 2 to 10 mM, the proportion of cells showing normal apoptotic features gradually decreased. Intriguingly, treatment with 10 mM EDS did not result in death for most cells and was marked by an absence of DNA laddering and ultrastructural features of apoptosis and necrosis. However, incubation with 20 mM EDS resulted in necrosis.These results demonstrated that the effects of EDS on cell survival are not specific to Leydig cells, that different cell types have different sensitivities to EDS and that stimulation of the cAMP pathway may mitigate EDS action. The data obtained with H540 cells further revealed that EDS can induce two types of programmed cell death. http://repub.eur.nl/res/pub/8360/ 2004-01-01T00:00:00Z The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active. http://repub.eur.nl/res/pub/3221/ 2004-01-01T00:00:00Z The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals. http://repub.eur.nl/res/pub/10083/ 2003-01-01T00:00:00Z The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes http://repub.eur.nl/res/pub/3207/ 2003-01-01T00:00:00Z The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes. http://repub.eur.nl/res/pub/8439/ 2003-01-01T00:00:00Z Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells. http://repub.eur.nl/res/pub/10010/ 2002-12-01T00:00:00Z Lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. FMRP is an RNA binding protein thought to be involved in translation efficiency and/or trafficking of certain mRNAs. Recently, a subset of mRNAs to which FMRP binds with high affinity has been identified. These FMRP-associated mRNAs contain an intramolecular G-quartet structure. In neurons, dendritic mRNAs are involved in local synthesis of proteins in response to synaptic activity, and this represents a mechanism for synaptic plasticity. To determine the role of FMRP in dendritic mRNA transport, we have generated a stably FMR1-enhanced green fluorescent protein (EGFP)-transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP-GFP fusion protein. After doxycycline induction, FMRP-GFP was localized in granules in the neurites of PC12 cells. By using time-lapse microscopy, the trafficking of FMRP-GFP granules into the neurites of living PC12 cells was demonstrated. Motile FMRP-GFP granules displayed two types of movements: oscillatory (bidirectional) and unidirectional anterograde. The average velocity of the granules was 0.19 micro m/s with a maximum speed of 0.71 micro m/s. In addition, we showed that the movement of FMRP-GFP labeled granules into the neurites was microtubule dependent. Colocalization studies further showed that the FMRP-GFP labeled granules also contained RNA, ribosomal subunits, kinesin heavy chain, and FXR1P molecules. This report is the first example of trafficking of RNA-containing granules with FMRP as a core constituent in living PC12 cells. http://repub.eur.nl/res/pub/17009/ 1998-05-27T00:00:00Z Reproduction is the most important factor for the maintenance of a species. The key event in this process Is fertilization: combination of haploid cells from male and female parents, spermatozoon and oocyte, respectively. Before spermatozoa and oocytes are formed and ready for fertilization many processes have taken place. which are very different for spermatozoa and oocytes. One of the most striking differences is the continuous production of newly formed spermatozoa in the fertile male, while the formation of oocytes is already complete before birth. The oocyte is surrounded by supporting cells. In the complex of the oocyte with the surrounding cells, called the follicle, close collaboration exists between the oocyte and the surrounding cells. The main part of this thesis describes the dynamics of follicle development and the hormonal factors influencing this development. http://repub.eur.nl/res/pub/8658/ 1997-01-01T00:00:00Z The objective of this study was to set up a superovulation protocol in adult cyclic rats by using recombinant human follicle stimulating hormone (rhFSH; Org32489). Good results were obtained by treatment with decreasing doses of rhFSH (2.5 to 0.5 IU) during the dioestrus period. The number of corpora lutea (CL) found in rats treated with this protocol was 43.5 +/- 3.4; this is more than three times the number in saline-treated control rats (13.0 +/- 0.4). Fertilization of oocytes after superovulation was as good as after normal ovulation in terms of number of 2-cell stage embryos found 2 days after mating. The absolute number of implantations was twice the number observed in saline-treated control rats (23.3 +/- 1.8 versus 10.6 +/- 0.5); therefore the number of implantations per CL was lower in superovulated rats. The serum concentrations of luteinizing hormone (LH), endogenous FSH and oestradiol-17beta were decreased during rhFSH treatment, while the inhibin serum concentration was increased. The progesterone serum concentration was increased on the days of pro-oestrus and oestrus after treatment. No difference was observed in the testosterone serum concentration. Pretreatment with 10 IU rhFSH at oestrus before giving the decreasing doses of rhFSH during dioestrus reduced the ovulatory response. Finally, treatment with a constant low dose of rhFSH instead of a decreasing dose of rhFSH did not result in spontaneous ovulation. However, ovulation induction by means of a human chorionic gonadotrophin bolus resulted in superovulation in six out of eight rats. It is concluded that superovulation in cyclic rats can be achieved using rhFSH treatment. However, it was found that the type of rhFSH regimen was very important to achieve appropriate stimulation. The optimal protocol was treatment with decreasing doses of rhFSH during dioestrus. The oocytes retrieved could be fertilized as well as oocytes of saline-treated control rats. The results also indicate that treatment with higher doses of rhFSH might induce a desensitization for FSH and LH.