Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase : Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (λ, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4–15 h) immunofluorescent labelling was restricted to a small part of the nucleus ( , 4.5 % of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle

MODELLING THE INFLUENCE OF NUCLEUS ELASTICITY ON CELL INVASION IN FIBER NETWORKS AND MICROCHANNELS

Cell migration in highly constrained extracellular matrices is exploited in scaffold-based tissue engineering and is fundamental in a wide variety of physiological and pathological phenomena, among others in cancer invasion and development. Research into the critical processes involved in cell migration has mainly focused on cell adhesion and proteolytic degradation of the external environment. However, rising evidence has recently shown that a number of cell-derived biophysical and mechanical parameters, among others nucleus stiffness and cell deformability, plays a major role in cell motility, especially in the ameboid-like migration mode in 3D confined tissue structures. We here present an extended cellular Potts model (CPM) first used to simulate a micro-fabricated migration chip, which tests the active invasive behavior of cancer cells into narrow channels. As distinct features of our approach, cells are modeled as compartmentalized discrete objects, differentiated in the nucleus and in the cytosolic region, while the migration chamber is composed of channels of different widths. We find that cell motile phenotype and velocity in open spaces (i.e., 2D flat surfaces or large channels) are not significantly influenced by cell elastic properties. On the contrary, the migratory behavior of cells within subcellular and subnuclear structures strongly relies on the deformability of the cytosol and of the nuclear cluster, respectively. Further, we characterize two migration dynamics: a stepwise way, characterized by fluctuations in cell length, within channels smaller than nucleus dimensions and a smooth sliding (i.e., maintaining constant cell length) behavior within channels larger than the nuclear cluster. These resulting observations are then extended looking at cell migration in an artificial fiber network, which mimics cell invasion in a 3D extracellular matrix. In particular, in this case, we analyze the effect of variations in elasticity of the nucleus on cell movement. In order to summarize, with our simulated migration assays, we demonstrate that the dimensionality of the environment strongly affects the migration phenotype and we suggest that the cytoskeletal and nuclear elastic characteristics correlate with the tumor cell's invasive potentia

The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalises with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasise differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite

A role for the cleaved cytoplasmic domain of E-cadherin in the nucleus

Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by γ-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus

Modelling diffusional transport in the interphase cell nucleus

In this paper a lattice model for diffusional transport of particles in the interphase cell nucleus is proposed. Dense networks of chromatin fibers are created by three different methods: randomly distributed, non-interconnected obstacles, a random walk chain model, and a self avoiding random walk chain model with persistence length. By comparing a discrete and a continuous version of the random walk chain model, we demonstrate that lattice discretization does not alter particle diffusion. The influence of the 3D geometry of the fiber network on the particle diffusion is investigated in detail, while varying occupation volume, chain length, persistence length and walker size. It is shown that adjacency of the monomers, the excluded volume effect incorporated in the self avoiding random walk model, and, to a lesser extent, the persistence length, affect particle diffusion. It is demonstrated how the introduction of the effective chain occupancy, which is a convolution of the geometric chain volume with the walker size, eliminates the conformational effects of the network on the diffusion, i.e., when plotting the diffusion coefficient as a function of the effective chain volume, the data fall onto a master curve.Comment: 9 pages, 8 figure

Numerical simulations of elastic capsules with nucleus in shear flow

The shear-induced deformation of a capsule with a stiff nucleus, a model of eukaryotic cells, is studied numerically. The membrane of the cell and of its nucleus are modelled as a thin and impermeable elastic material obeying a Neo-Hookean constitutive law. The membranes are discretised by a Lagrangian mesh and their governing equations are solved in spectral space using spherical harmonics, while the fluid equations are solved on a staggered grid using a second-order finite differences scheme. The fluid-structure coupling is obtained using an immersed boundary method. The numerical approach is presented and validated for the case of a single capsule in a shear flow. The variations induced by the presence of the nucleus on the cell deformation are investigated when varying the viscosity ratio between the inner and outer fluids, the membrane elasticity and its bending stiffness. The deformation of the eukaryotic cell is smaller than that of the prokaryotic one. The reduction in deformation increases for larger values of the capillary number. The eukaryotic cell remains thicker in its middle part compared to the prokaryotic one, thus making it less flexible to pass through narrow capillaries. For a viscosity ratio of 5, the deformation of the cell is smaller than in the case of uniform viscosity. In addition, for non-zero bending stiffness of the membrane, the deformation decreases and the shape is closer to an ellipsoid. Finally, we compare the results obtained modeling the nucleus as an inner stiffer membrane with those obtained using a rigid particle

In Vivo Localization of Fas-Associated Death Domain Protein in the Nucleus and Cytoplasm of Normal Thyroid and Liver Cells

FADD (Fas-associated death domain) is the main death receptor adaptor molecule that transmits apoptotic signal. Recently, FADD protein was shown to be expressed both in the cytoplasm and nucleus of in vitro cell lines. In contrast to the cytoplasmic FADD, the nuclear FADD was shown to protect cells from apoptosis. However, in vivo subcellular localization of FADD was still unknown. Here, we demonstrated that FADD protein was expressed in both cytoplasmic and nuclear compartment in ex vivo thyroid cells demonstrating that nuclear sublocalization of FADD protein was a relevant phenomenon occurring in vivo. Moreover, we showed that in the nucleus of untransformed thyroid cells FADD localized mainly on euchromatin. We confirmed the nuclear localization of FADD in ex vivo liver and showed that in this organ FADD and MBD4 interact together. These results demonstrate that FADD is physiologically expressed in the nucleus of cells in at least two mouse organs. This particular localization opens new possible role of FADD in vivo either asan inhibitor of cell death, or as a transcription factor, or as a molecular link between apoptosis and genome surveillance

Genomic function during the lampbrush chromosome stage of amphibian oogenesis

Throughout its lengthy developmental history the disposition of the genetic material in the amphibian oocyte nucleus differs from that in other cell types. The chromosomes in the oocyte nucleus, arrested for the whole of oogenesis at the prophase of the first meiotic division, are known to contain at least the tetraploid amount of DNA.(1,2) Oogenesis in amphibia requires months or even years to complete, depending on the species