Wiggert A. van Cappellen

In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium

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.

Nuclear dynamics of PCNA in DNA replication and repair.

ABSTRACT 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.

Tracking in cell and developmental biology.

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.

Activation of multiple DNA repair pathways by sub-nuclear damage induction methods

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 photo-lesions allowing the study of the UV-C-induced DNA damage response in a user-defined area in cultured cells.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery

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.

The Ubiquitin-Conjugating DNA Repair Enzyme HR6A Is a Maternal Factor Essential for Early Embryonic Development in Mice

ABSTRACT 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.

Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends.

Microtubule (MT) plus end–tracking proteins (+TIPs) specifically 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 specificity 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 affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding 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 affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

Enhanced Chromatin Dynamics by FACT Promotes Transcriptional Restart after UV-Induced DNA Damage

Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response.

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH.

ABSTRACT The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

Stepwise androgen receptor dimerization

Androgen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding and DNA binding, and by specific AR protein–protein interactions. The latter include DNA-binding domain (D-box) interactions in AR homodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-binding domain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependent transition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization are initiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated by AR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra- and inter-molecular interactions.