Emerin binding to Btf, a death-promoting transcriptional repressor, is disrupted by a missense mutation that causes Emery-Dreifuss muscular dystrophy.

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 Delta95-99, disrupted binding to Btf. The Delta95-99 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.

Spectral imaging fluorescence microscopy.

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).

Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.