Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation.

The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.

A contractile nuclear actin network drives chromosome congression in oocytes.

Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.

4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracks.

BACKGROUND: The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. RESULTS: We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. CONCLUSIONS: Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

Nuclear body movement is determined by chromatin accessibility and dynamics.

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.