Nuclear-Cytoplasmic Trafficking of NTF2, the Nuclear Import Receptor for the RanGTPase, Is Subjected to Regulation

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).     

Quantitative fluorescence imaging of protein diffusion and interaction in living cells

Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination that allows massively parallel fluorescence correlation spectroscopy (FCS) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1α (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1α-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.     

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.     

The nuclear pore and plant development

All macromolecules that traffic between the nucleus and the cytoplasm traverse the nuclear pore. While yeast and mammalian nuclear pore structure and function have recently been substantially refined, our understanding of the plant nuclear pore is still far from comprehensive. Nevertheless, a number of nuclear pore and nucleocytoplasmic trafficking components have recently been identified as required for diverse developmental and signaling pathways. In addition, some aspects of the nuclear pore composition itself now appear under developmental control and nuclear pore components have recently surfaced as novel players in plant cytokinesis. Here, we review these new findings in context and attempt to correlate molecular functions with developmental processes.     

Efficient extraction of nucleolar proteins for interactome analyses

The efficient extraction of proteins from purified cellular organelles is critical for in vitro analyses, including identification of protein complex members by affinity purification‐based quantitative proteomic approaches. When applied to purified nucleoli, classic nuclear protein extraction methods inefficiently and selectively release only ～50% of proteins. Here, we present a method that can extract up to 90% of nucleolar proteins, and apply it in a quantitative interactomic approach to identify nucleolar interaction partners for a mammalian protein phosphatase.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Reshaping of the endoplasmic reticulum limits the rate for nuclear envelope formation

During mitosis in metazoans, segregated chromosomes become enclosed by the nuclear envelope (NE), a double membrane that is continuous with the endoplasmic reticulum (ER). Recent in vitro data suggest that NE formation occurs by chromatin-mediated reorganization of the tubular ER; however, the basic principles of such a membrane-reshaping process remain uncharacterized. Here, we present a quantitative analysis of nuclear membrane assembly in mammalian cells using time-lapse microscopy. From the initial recruitment of ER tubules to chromatin, the formation of a membrane-enclosed, transport-competent nucleus occurs within ~12 min. Overexpression of the ER tubule-forming proteins reticulon 3, reticulon 4, and DP1 inhibits NE formation and nuclear expansion, whereas their knockdown accelerates nuclear assembly. This suggests that the transition from membrane tubules to sheets is rate-limiting for nuclear assembly. Our results provide evidence that ER-shaping proteins are directly involved in the reconstruction of the nuclear compartment and that morphological restructuring of the ER is the principal mechanism of NE formation in vivo.     

Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.     

Mammalian sirtuins and energy metabolism

Sirtuins are highly conserved NAD+-dependent protein deacetylases and/or ADP-ribosyltransferases that can extend the lifespan of several lower model organisms including yeast, worms and flies. The seven mammalian sirtuins, SIRT1 to SIRT7, have emerged as key metabolic sensors that directly link environmental signals to mammalian metabolic homeostasis and stress response. Recent studies have shed light on the critical roles of sirtuins in mammalian energy metabolism in response to nutrient signals. This review focuses on the involvement of two nuclear sirtuins, SIRT1 and SIRT6, and three mitochondrial sirtuins, SIRT3, SIRT4, and SIRT5, in regulation of diverse metabolic processes.     

Uptake of endocytic markers into mitotic yeast cells

[3H]alpha-Factor and Lucifer yellow were used to measure receptor mediated and fluid-phase endocytosis in the yeast Saccharomyces cerevisiae, arrested in mitosis by depolymerization of the microtubules or due to a mutation preventing nuclear division (cdc16). Both processes continued at roughly the same level as during interphase. This shows that in yeast endocytosis is not interrupted during mitosis like in mammalian cells.     

Coherent activation of a synthetic mammalian gene network

A quantitative analysis of naturally-occurring regulatory networks, especially those present in mammalian cells, is difficult due to their high complexity. Much simpler gene networks can be engineered in model organisms and analyzed as isolated regulatory modules. Recently, several synthetic networks have been constructed in mammalian systems. However, most of these engineered mammalian networks have been characterized using steady-state population level measurements. Here, we use an integrated experimental-computational approach to analyze the dynamical response of a synthetic positive feedback network in individual mammalian cells. We observe a switch-like activation of the network with variable delay times in individual cells. In agreement with a stochastic model of the network, we find that increasing the strength of the positive feedback results in a decrease in the mean delay time and a more coherent activation of individual cells. Our results are important for gaining insight into biological processes which rely on positive feedback regulation.