The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.

Lake Vostok, the 7th largest (by volume) and 4th deepest lake on Earth, is covered by more than 3,700 m of ice, making it the largest subglacial lake known. The combination of cold, heat (from possible hydrothermal activity), pressure (from the overriding glacier), limited nutrients and complete darkness presents extreme challenges to life. Here, we report metagenomic/metatranscriptomic sequence analyses from four accretion ice sections from the Vostok 5G ice core. Two sections accreted in the vicinity of an embayment on the southwestern end of the lake, and the other two represented part of the southern main basin. We obtained 3,507 unique gene sequences from concentrates of 500 ml of 0.22 µm-filtered accretion ice meltwater. Taxonomic classifications (to genus and/or species) were possible for 1,623 of the sequences. Species determinations in combination with mRNA gene sequence results allowed deduction of the metabolic pathways represented in the accretion ice and, by extension, in the lake. Approximately 94% of the sequences were from Bacteria and 6% were from Eukarya. Only two sequences were from Archaea. In general, the taxa were similar to organisms previously described from lakes, brackish water, marine environments, soil, glaciers, ice, lake sediments, deep-sea sediments, deep-sea thermal vents, animals and plants. Sequences from aerobic, anaerobic, psychrophilic, thermophilic, halophilic, alkaliphilic, acidophilic, desiccation-resistant, autotrophic and heterotrophic organisms were present, including a number from multicellular eukaryotes.

Bacteria from seven Lake Vostok accretion and two deep glacial Vostok ice core sections were characterized. The cell concentrations were low, but many of the cells were viable. From the hundreds of cultures, 18 unique bacterial rRNA gene phylotypes were determined. Lake Vostok may contain a complex microbial ecosystem.Subglacial Lake Vostok, the eighth largest lake on Earth (area ϭ 14,000 km 2 , volume ϭ 5,600 km 3 ) (9, 16), is covered by a 4-kilometer-thick layer of glacial ice. As the glacier traverses the lake over a period of 18,000 years, ice freezes (or accretes) to the bottom surface of the glacier, eventually forming a 200-m layer of accretion ice that has retained a linear and temporal record of the contents of the upper surface of the lake. The glacier passes over a shallow embayment, near an island (or peninsula), and then over part of the main lake basin. As the glacier passes through the embayment, initially it is grounded on the lakebed, and partly because of this, it collects mineral inclusions, making the ice silty (termed type I accretion ice) (13). Melting and freezing in this area, as well as a possible influx of material from a river system and/or from hydrothermal activity, may contribute to the characteristics of the type I ice (3, 14). The glacier is suspended over open water in portions of the embayment and over most of the main parts of the lake. The ice that forms over open water contains far fewer inclusions and lower concentrations of ions, organic carbon, and biomass (6,10,14). This ice is very clear ice and has been termed type II accretion ice (13). The top section (from 3,538 to 3,595 m), which accreted within and near the embayment, primarily consists of type I ice, although there are some regions of type II ice (2). Bacteria from this ice, including potentially psychrophilic and psychrotolerant species as well as the molecular signature of a thermophilic bacterium, have been reported (1,3,4,5,7,10). Within the lake, temperatures average Ϫ2°C, pressures approach 400 atmospheres, high oxygen levels exist, there are low nutrient levels, and it is completely dark.We isolated and characterized microbes from Lake Vostok type I and II accretion ice from the embayment and the main basin as well as from glacial ice immediately above the accretion ice layers. Sequence results from the rRNA small subunit genes and internal transcribed spacers indicate that at least 18 species are represented in the accretion ice. All are psychrotolerant in that they grew at 4°C, although optimal growth was often at higher temperatures.Descriptions and cell concentrations. Nine Vostok 5G ice core sections were assayed. Five sections represented ice that accreted over the shallow embayment (depths of 3,540, 3,563, 3,582, and 3,584 m, all type I ice, and 3,591 m, type II ice) (2) approximately 3,800 to 5,100 years ago (2,6,13,14), two accreted 3,400 to 3,500 years ago over the main lake basin (3,606 m, type I ice, and 3,610 m, type II ice), and two were glacial ice cores near the bottom of the glacier (3,5...

Here we report the characterization of fungi from 10 accretion ice sections (3300-5100 y old) as well as two deep glacial ice sections that are close to the bottom of the glacier (1,000,000-2,000,000 y old) from the Vostok, Antarctica, 5G ice core. Fungi were characterized by fluorescence microscopy culturing and sequence analyses of ribosomal DNA internal transcribed spacers. A total of 270 fungal colonies were cultured from the accretion ice of subglacial Lake Vostok and an additional 14 from the glacial ice immediately above the accretion ice. Mean concentrations were 0-4.42 cells mL(-1) ice meltwater of which 0-100% exhibited viability (as determined by fluorescence microscopy). Thirty-one unique fungal ribosomal DNA sequences (28 from accretion ice and three from glacial ice) were determined and compared to recent taxa. The results, plus tests for growth at low temperatures, indicated that Lake Vostok contains a mixture of heterotrophic psychrotolerant fungal species. This indicates that the lake is not sterile but contains a unique ecosystem.

Ice deposited in Greenland and Antarctica entraps viable and nonviable microbes, as well as biomolecules, that become temporal atmospheric records. Five sections (estimated to be 500, 10,500, 57,000, 105,000 and 157,000 years before present, ybp) from the GISP2D (Greenland) ice core, three sections (500, 30,000 and 70,000 ybp) from the Byrd ice core, and four sections from the Vostok 5G (Antarctica) ice core (10,500, 57,000, 105,000 and 105,000 ybp) were studied by scanning electron microscopy, cultivation and rRNA gene sequencing. Bacterial and fungal isolates were recovered from 10 of the 12 sections. The highest numbers of isolates were found in ice core sections that were deposited during times of low atmospheric CO2, low global temperatures and low levels of atmospheric dust. Two of the sections (GISP2D at 10,500 and 157,000 ybp) also were examined using metagenomic/metatranscriptomic methods. These results indicated that sequences from microbes common to arid and saline soils were deposited in the ice during a time of low temperature, low atmospheric CO2 and high dust levels. Members of Firmicutes and Cyanobacteria were the most prevalent bacteria, while Rhodotorula species were the most common eukaryotic representatives. Isolates of Bacillus, Rhodotorula, Alternaria and members of the Davidiellaceae were isolated from both Greenland and Antarctica sections of the same age, although the sequences differed between the two polar regions.