Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments.

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Compartmentalization of regulatory proteins in the cell nucleus.

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.

Rapid exchange of histone H1.1 on chromatin in living human cells.

The considerable length of DNA in eukaryotic genomes requires packaging into chromatin to fit inside the small dimensions of the cell nucleus. Histone H1 functions in the compaction of chromatin into higher order structures derived from the repeating 'beads on a string' nucleosome polymer. Modulation of H1 binding activity is thought to be an important step in the potentiation/depotentiation of chromatin structure for transcription. It is generally accepted that H1 binds less tightly than other histones to DNA in chromatin and can readily exchange in living cells. Fusion proteins of Histone H1 and green fluorescent protein (GFP) have been shown to associate with chromatin in an apparently identical fashion to native histone H1. This provides a means by which to study histone H1-chromatin interactions in living cells. Here we have used human cells with a stably integrated H1.1-GFP fusion protein to monitor histone H1 movement directly by fluorescence recovery after photobleaching in living cells. We find that exchange is rapid in both condensed and decondensed chromatin, occurs throughout the cell cycle, and does not require fibre-fibre interactions. Treatment with drugs that alter protein phosphorylation significantly reduces exchange rates. Our results show that histone H1 exchange in vivo is rapid, occurs through a soluble intermediate, and is modulated by the phosphorylation of a protein or proteins as yet to be determined.