Science and research

The Institute of Cellular Biology and Pathology (ICBP) is primarily engaged in systematic studies of the cell nucleus of (mainly) mammalian cells. Its long-standing interests are primarily:

• (large-scale) chromatin organization
• integration of the processes of DNA replication and RNA synthesis/processing in the context of a functional nuclear architecture
• structure of the chromatin fiber

The scientific projects are mostly based on genetic, biochemical, molecular biological and cell biological approaches complemented by advanced methods in live cell imaging, electron microscopy, correlative light and electron microscopy and (cryo-) electron tomography. Also, for structural biology and nanotechnology projects, native cryo-electron microscopy has been implemented to analyze (nucleo)protein complexes and nanotools (e.g. nanocapsules). The development and utilization of a new generation programmable array microscope (PAM) is also carried out. The Laboratory of Cell Biology has been a joint venture laboratory of the ICBP of the First Faculty of Medicine at Charles University in Prague and the Department of Cell Biology of the Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i.

Functional organization of the cell nucleus

Large-scale chromatin organization

• By fluorescent labelling of selected chromatin regions in living HepG2 cells expressing recombinant histone H4-Dendra2, we investigated the extent to which the chromatin arrangement of mother cells is maintained in daughter cells. We have demonstrated that, compared to the mother cell, the distribution of photoconverted chromatin areas was significantly different in daughter cells, though not totally random (Cvackova et al., 2009).
• Evidence was presented for the reversible, cold-induced immunodetection of a nuclear epitope (referred to as epiC) recognized by a monoclonal anti-actin antibody. The temporal and spatial properties of epiC labelling suggest that it constitutes a modification of the early S phase chromatin domains that persists until the early G1 phase of the next cell cycle. The epiC marker has been identified as a dual post-translational modification of histone H4 and apparently has an important role in the transfer/maintenance of epigenetic information on the transcriptionally-competent part of the genome (Fidlerova et al., 2009).
• In collaboration with the University of Buffalo, New York, USA (Prof. R. Berezney) we have deciphered the organization of the interferon (IFN) gene cluster in a human tumour cell line using a combination of FISH and advanced chomosomal analysis. We studied the arrangement of the amplified IFN gene cluster as well as that of associated segments of chromosomes and centromeric DNA repeats in the interphase cell nucleus. We also presented a model for the generation of the rearranged chromosome through breakage-fusion-bridging events (Marella et al., 2008; Zeitz et al., 2009).
• Replication protein A (RPA) is involved in DNA replication, repair and recombination. Using restriction endonucleases, we induced double-strand (ds) breaks in the DNA of sperm nuclei formed in Xenopus laevis egg extracts. We observed the formation of foci enriched in RPA and Ku protein after limited digestion. Additional biochemical, molecular biology, ultrastructural, immunocytochemical and in situ hybridization experiments proved that the nuclei reconstituted in vitro from Xenopus laevis extract represent a suitable model for studying the repair mechanisms of dsDNA breaks (Eltsov et al., 2000; Grandi et al., 2001).
• In collaboration with the Institute of Biophysics of the Czech Academy of Sciences we have analyzed histone H3-Lys9 acetylation (H3K9), an epigenetic mark associated with transcriptionally active chromatin, during endoderm-like differentiation of human embryonic stem cells. Using genome wide ChIP-on-chip analysis we have shown that out of 24,659 promoters only 117 H3K9-acetylated promoters were involved in pluripotency and 25 were responsible for endoderm-like differentiation. The analysis of the levels of H3K9 acetylation suggested that chromosomes 11, 12, 17 and 19 were critical both for pluripotency and endoderm-like differentiation (Krejci et al., 2009).
• As part of the same collaboration, we have found that lamin A/C deficiency results in condensation of chromosomal territories and reorganization of centromeric heterochromatin. The inhibition of histone-deacetylase reversed these changes to a large extent, suggesting that the interaction of lamins A/C with specific histone modifications can play an important role in higher level chromatin organization (Galiova et al., 2008).
• In respect to understand the formation of large scale chromatin structures at the nuclear level, we studied also the Polycomb mediated gene silencing by microscopical approaches. So far, we published (Smigova et al., 2011) our results on the identification of the "PcG body." We found out the identity of the "PcG body" as the locally higher spatial accumulation of heterochromatin structures not bearing the characteristics of a nuclear body or silencing factory. According to our results, we designed a model of the PcG domain that could help to understand the gene silencing at the nuclear level.

Structure-function correlates of DNA replication

It is known that DNA replication in mammalian cells is initiated simultaneously at many replication foci. The spatial localization of these foci in the cell nucleus vary during S phase, as does the speed of the replication process.

• We have described the dynamics of actively replicating chromatin domains (Masata et al., 2005), determined the speed of the replication fork, and found differences in the rate of replication between early and late S phase (Malinsky et al., 2001).
• Using electron microscopy, we identified actively replicating chromatin structures of uniform size which represent the basic functional units of replicating chromatin (Koberna et al., 2004).
• In vivo labelling of DNA segments in combination with electron tomography allowed us to suggest a new model for the organization of replicated DNA. According to this model, replisome couples produce loops with the associated arms in the form of four tightly associated 30nm fibers (Ligasova et al., 2009).

Reorganization of chromatin as a result of transcription and replication of ribosomal genes

Human ribosomal genes are organized as tandem repeats on five pairs of chromosomes (13, 14, 15, 21 and 22) in regions called Nucleolar Organizing Regions (NORs). The diploid genome contains around 400 ribosomal genes, but, importantly, not all of these genes are transcriptionally active. Although transcription of rDNA is blocked during mitosis, it has been recognized that the potential for certain genes to be transcribed is maintained until the next cell cycle. Moreover, transcription of active ribosomal genes during interphase is: i) located exclusively in the nucleolus, which is visible by phase contrast microscopy and ii) can be specifically modulated. This makes the ribosomal genes an ideal model for investigating the impact of both transcription and replication on the organization of chromatin in the nucleus.

• We demonstrated that the transcriptional activity of rDNA in HeLa cells is regulated at the level of functional units containing several genes (Pliss et al., 2005), and that transcriptionally active genes are situated in the dense fibrillar components of the nucleolus (Koberna et al., 2002; Malinsky et al., 2002).
• We also studied the transcriptional activity of NORs during the cell cycle and the association of NOR-bearing chromosomes with the nucleoli. Nucleolar association of these chromosomes correlated with the transcriptional activity of the corresponding NORs (Kalmarova et al, 2007). Although daughter cells contained a different number of nucleoli when compared to the mother cell, the nucleolar associations of the chromosomes that were studied showed more similarity in daughter cells than in unrelated cells (Smirnov et al., 2006; Kalmarova et al., 2008a). In vivo study of NORs in cells expressing UBF-GFP showed that transcriptionally-competent NORs are unequally distributed between the daughter cells in mitosis (Kalmarova et al., 2008b).
• In the framework of a collaboration with Prof. I. Grummt at the German Cancer Research Centre in Heidelberg, we contributed to the finding that the NoRC is an important determinant of replication timing and that epigenetic marks are heritably maintained through DNA replication (Li et al., 2005).
• We have detected Pontin in nucleoli, particularly in nucleolar fibrillar centers. Through functional experiments we uncovered a role for Pontin in the regulation of c-myc dependent rRNA synthesis (Cvackova et al., 2008).
• In a collaboration with the laboratory of Dr. J. McNally (NIH, Bethesda) we demonstrated the potential role of ubiquitin and the proteasome in the biogenesis of ribosomes (Stavreva et al., 2006).
• We wrote three reviews that summarize the current state of knowledge about the nucleolus. We focused on the way in which the biochemical processes that are known to take place in the nucleolus correlate with nucleolar structure as seen both by light and electron microscopy (Raska et al., 2006a, b; Cmarko et al., 2008).

Splicing of pre-mRNA

Factors involved in pre-RNA splicing accumulate in nuclear speckles.

• We hypothesized that speckles are involved in splicing (Melcak and Raska, 1996) and, in support of this hypothesis, found bidirectional intranuclear movement of RNA transcripts and splicing factors, recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors (nuclear speckles) (Melcak et al., 2000).
• We identified nuclear speckles as the compartment where splicing complexes are assembled (Melcak et al., 2001; Kopsky et al., 2002).
• We also addressed the problem of speckle formation and stability (Vecerova et al., 2004). We showed that both the formation and the dynamics of the nuclear speckles do not depend on the presence of lamins A/C.
• We reviewed the current data related to the function of the Cajal body, with an emphasis on the role of this nuclear body in the assembly of snRNPs (Stanek and Neugebauer, 2006).

Nucleosome structure, structure of the basic nucleosomal particle containing histone variants and nucleosome remodeling using chromatin remodeling complexes

• In collaboration with Dr. S. Dimitrov (Institut Albert Bonniot, Grenoble, France), we have succeeded in performing the biochemical and structural characterization of a nucleosome containing the histone variant H2A. The results, which were obtained using atomic force microscopy (AFM) and cryo-electron microscopy, show that the histone octamers containing this histone variant associate with only about 130 DNA base pairs (about 10 base pairs from each nucleosome are dissociated from the octamer). This configuration suggests a lower stability for nucleosomes containing this histone variant (Doyen et al., 2006). Using a similar approach, we have found that nucleosomes containing the histone variant H2A.L2 - the mouse testis-specific variant of histone H2A - associate with about 20 bp less than conventional nucleosomes, resulting in a more open structure (Syed et al, 2009).
• Using cryo-electron microscopy, we have also characterized nucleosomes to which linker histones were added using the NAP-1 complex. We have demonstrated that the NAP-1-mediated association of the linker histone is very efficient. Furthermore, we showed that the NAP-1 complex is also capable of mediating the association of linker histone mutants and of its isolated globular domain.
• Studies of the remodeling of nuclesomes by the RSC chromatin remodeling complex revealed the existence of a metastable remodeling product - a nucleosome with a modified structure. We showed that such nucleosomes incorporate more DNA (20 to 40 bp). The excess of DNA in the particle leads to the dissociation of the DNA from the histone octamer and consequently to a deformation of the circular shape of the nucleosome. It also results in particles displaying an increased accessibility to nucleosomal DNA when compared to intact particles.

Morphogenesis of the genome during early embryogenesis of C. elegans

The main goal of this project is to analyze the morphogenesis of the genome in the cell nuclei of early Caenorhabditis elegans embryos in an attempt to uncover functional links with developmental potential and cell differentiation. The morphogenesis of the genome is the process whereby linear genetic information folds into a complex three-dimensional (3D) shape in the nucleus.

The adult C. elegans hermaphrodite is made up of only 959 cells (558 at the end of embryonic development approx. 14h post-fertilization) and the entire cell lineage has been determined. C. elegans develops according to an invariant cell lineage, with constant cell position and synchronous cell division from individual to individual. This developmental constancy is the most relevant property for the present proposal, as it will allow comparing gene positioning in cells that are rigorously equivalent in terms of history, developmental potential and gene expression profile. The positioning of genes in the C. elegans embryonic cell nuclei is determined using multicolor 3D DNA FISH and live cell imaging. The finding of reproducible patterns of spatial gene positioning during early embryogenesis would open new avenues of research on genome organization and gene regulation.

New technologies and nanotechnologies

Development and utilization of a new generation programmable array microscope (PAM)

We are also focusing on development of a unique confocal microscope. This microscope (the programmable array microscope, or PAM) has a performance exceeding commercially available designs. It will be able to create diverse illumination patterns by spatial light modulators, which are faster and more easily controllable than conventional piezo-driven diffraction grids.

The PAM will be utilized in two kinds of experiments:

1. 4D monitoring of dynamic cellular events. We will use transfected cells synthetizing recombinant proteins with photo-activable or photo-convertible fluorescent proteins (see e.g. Cvarkova et al., 2009). We suppose that PAM will be ~ 10x more sensitive than conventional scanning confocal microscopes, which will allow significant decrease of the light dose necessary for imaging (Hagen et al., 2007). This will lead to reduction of the well known accompanying hampering effects - phototoxicity and photobleaching - which will in turn allow monitoring of cellular phenomena more often and in longer time periods.
2. Optical sectioning and high-resolution imaging by structured-illumination microscopy (SIM). SIM combines microscopic and computational approaches in order to reach a two-fold increase of the conventional light microscopy resolution set by the Abbe diffraction limit.

The current version of the PAM was used to measure lateral diffusion of erbB3-mCitrine molecules. The measurement was performed both in control cells and in experiment cells, which were treated by substances affecting cytoskeleton, plasmatic membranes or activate the co-expressed erbB1 (Hagen et al., 2009).

The final construction of the PAM will bring us a highly sensitive microscope for real-time high-resolution imaging of dynamic cellular phenomena, in particular at studies of cell nucleus.

Single-molecule localization microscopy (SMLM)

Single-molecule localization microscopy is a new and very effective kind of light microscopy, which enables imaging of fluorescent samples with resolution far beyond the Abbe diffraction limit. The method localizes fluorescent molecules that are forced to "blinking" by a strong beam of light, in which the fluorescent molecules change their non-emissive state into emissive state. The emission is a strong flash that is computationally localized. The only requirement for this method is sufficiently intensive illumination of the fluorescent specimen. Originally, this method was called PALM (Photoactivated localization microscopy) and it could be used only with the Dronpa fluorescent protein. Later it was found that also GFP, Dendra2 and EOS were suitable for PALM.

At ICBP we have been already able to monitor expression of erbB3-mCitrine in A431 cells. This gene is expressed or at least present in mutant forms of some types of malignant tumours, in particular breast and brain tumours. The superresolution of our imaging was 20 nm, which is about ten-fold higher resolution compared to conventional light microscopy.

Nanotechnologies

• Using cryo-electron microscopy, we have analyzed the morphology of hybrid silicon-silica nanocapsules prepared by template formation (Kepczynski et al., 2009, 2010). Their properties suggest they could be used as vectors for targeted drug delivery in organisms.
• In a collaboration with the Faculty of Sciences at J.E. Purkyne University in Usti nad Labem, we have studied structural, optical and electrochemical properties of thin nanocomposite Sn/hydrocarbon plasma polymer films. We found by electron tomography that, unlike similar nanocomposite materials composed of small amounts of metals, the Sn is irregularly distributed within the polymer layer (Matousek et al., 2009). We have also found that the basic physical properties of the materials strongly depend on the sputtering conditions.
• In another collaboration with the Faculty of Sciences at J.E. Purkyne University in Usti nad Labem, we have analyzed newly-synthesized nanoparticle labels having bioactive properties by transmission electron microscopy. These biotinylated labels are formed on silver nanoparticles embedded in a basic polymer matrix made of PAMAM dendrimers. The diameters of the silver nanoparticles correspond to the specific spatial organization of the polymer matrix. The activity of biotin after synthesis was successfully demonstrated through immunolabeling tests (Maly et al., 2009).