Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct `core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.

Loss of functional emerin, a nuclear membrane protein, causes X‐linked recessive Emery–Dreifuss muscular dystrophy. In a yeast two‐hybrid screen, we found that emerin interacts with Btf, a death‐promoting transcriptional repressor, which is expressed at high levels in skeletal muscle. Biochemical analysis showed that emerin binds Btf with an equilibrium affinity (KD) of 100 nm. Using a collection of 21 clustered alanine‐substitution mutations in emerin, the residues required for binding to Btf mapped to two regions of emerin that flank its lamin‐binding domain. Two disease‐causing mutations in emerin, S54F and Δ95–99, disrupted binding to Btf. The Δ95–99 mutation was relatively uninformative, as this mutation also disrupts emerin binding to lamin A and a different transcription repressor named germ cell‐less (GCL). In striking contrast, emerin mutant S54F, which binds normally to barrier‐to‐autointegration factor, lamin A and GCL, selectively disrupted emerin binding to Btf. We localized endogenous Btf in HeLa cells by indirect immunoflurorescence using affinity‐purified antibodies against Btf. In nonapoptotic HeLa cells Btf was found in dot‐like structures throughout the nuclear interior. However, within 3 h after treating cells with Fas antibody to induce apoptosis, the distribution of Btf changed, and Btf concentrated in a distinct zone near the nuclear envelope. These results suggest that Btf localization is regulated by apoptotic signals, and that loss of emerin binding to Btf may be relevant to muscle wasting in Emery–Dreifuss muscular dystrophy.

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

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The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).Since the discovery of the jellyfish green fluorescent protein (GFP) and its colour variants, multiple wavelength fluorescence imaging has become a useful tool in studies of cell biology. For imaging multiple wavelength fluorescence images, computer-controlled microscope systems that automatically switch optical filters during observation have been developed (Hiraoka et al . 1991;Haraguchi et al . 1999). While such microscope systems can collect fluorescence images at multiple wavelengths, fluorescence emissions with a spectral overlap can cause cross-talk between the emitted signals, thus limiting the choice of fluorescent dyes.Here we introduce a microscope technique that is capable of resolving the spectra of fluorescence images without switching optical filters; the fluorescence spectra are obtained as a series of images as a function of fluorescence wavelength. This capability of spectral resolution makes it possible to unmix the cross-talk between overlapping fluoresence emissions. It also provides a unique capability for detecting spectral changes in fluorescent indicators; for example, an indicator for fluorescence resonance energy transfer (FRET). Such techniques have been devised by the use of an acoustooptic tunable filter (AOTF) or a grating (Wachman et al . 1997;Hanley et al . 2000;Tsurui et al . 2000;Ford et al . 2001;Lansford et al . 2001).The hardware design we used is an implementation of the previously reported technology of two-photon imaging spectroscopy (Lansford et al . 2001) to a commercial single-photon confocal scanning microscope; the technology is applicable to a single-photon or multiphoton fluorescence microscope. In this spectral imaging microscope system (LSM510 META; Carl Zeiss, Jena, Germany), fluorescence spectra are resolved using grating optics; resolved fluorescence spectra at each pixel are detected by an array of 32 photomultiplier tubes (PMT) placed at the confocal plane, and recorded on a pixel-by-pixel basis during scanning to generate a set of images, each corresponding to the fluorescence wavelength resolved at 10 nm intervals. The maximum range of wavelength is 380 -720 nm, resolved to 32 divisions. Spectral resolution of fluorescence imagesWe first demonstrate that the use of grating optics allows us to obtain multiple-wavelength images without switching optical filters. An example of spectral imaging, or imaging spectroscopy, is shown in Fig. 1A. ...

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF + DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.barrier-to-autointegration factor | DNA-bead | autophagy | nuclear envelope | DNA sensor

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to ∼80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.