Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.

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 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. ...

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Most mammalian cell strains genetically deficient in peroxisome biogenesis have abnormal membrane structures called ghosts, containing integral peroxisomal membrane protein, PMP70, but lacking the peroxisomal matrix proteins. Upon genetic complementation, these mutants regain the ability of peroxisome biogenesis. It is postulated that, in this process, the ghosts act as the precursors of peroxisomes, but there has been no evidence to support this. In the present study, we investigated this issue by protein microinjection to a mutant Chinese hamster ovary cell line defective of PEX5, encoding a peroxisome-targeting signal receptor. When recombinant Pex5p and green fluorescent protein (GFP) carrying a peroxisome-targeting signal were co-injected into the mutant cells, the GFP fluorescence gathered over time to particulate structures where PMP70 was co-localized. This process was dependent on both Pex5p and the targeting signal, and, most importantly, occurred even in the presence of cycloheximide, a protein synthesis inhibitor. These findings suggest that the ghosts act as acceptors of matrix proteins in the peroxisome recovery process at least in the PEX5 mutant, and support the view that peroxisomes can grow by incorporating newly synthesized matrix proteins.