Charles University, 1st Faculty of Medicine

Albertov 4, 12801 Praha 2, Czech Republic - tel.: +420 224 968 001, e-mail: lge @

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:

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

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.

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.

Splicing of pre-mRNA

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

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

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.