The human T-cell leukemia virus type 1 (HTLV-1) is the cause of adult T-cell leukemia/lymphoma as well as tropical spastic paraparesis/ HTLV-1-associated myelopathy. HTLV-1 is transmitted to T cells through the virological synapse and by extracellular viral assemblies. Here, we uncovered an additional mechanism of virus transmission that is regulated by the HTLV-1-encoded p8 protein. We found that the p8 protein, known to anergize T cells, is also able to increase T-cell contact through lymphocyte function-associated antigen-1 clustering. In addition, p8 augments the number and length of cellular conduits among T cells and is transferred to neighboring T cells through these conduits. p8, by establishing a T-cell network, enhances the envelope-dependent transmission of HTLV-1. Thus, the ability of p8 to simultaneously anergize and cluster T cells, together with its induction of cellular conduits, secures virus propagation while avoiding the host's immune surveillance. This work identifies p8 as a viral target for the development of therapeutic strategies that may limit the expansion of infected cells in HTLV-1 carriers and decrease HTLV-1-associated morbidity.human leukemia retrovirus | orf-I

The nuclear repositioning of specific genes may be a novel diagnostic strategy to distinguish malignant from normal tissue.

Chromosomes and genes are nonrandomly arranged within the mammalian cell nucleus. However, the functional significance of nuclear positioning in gene expression is unclear. Here we directly probed the relationship between nuclear positioning and gene activity by comparing the location of the active and inactive copies of a monoallelically expressed gene in single cell nuclei. We demonstrate that the astrocyte-specific marker GFAP (glial fibrillary acidic protein) is monoallelically expressed in cortical astrocytes. Selection of the active allele occurs in a stochastic manner and is generally maintained through cell division. Taking advantage of the monoallelic expression of GFAP, we show that the functionally distinct alleles occupy differential radial positions within the cell nucleus and differentially associate with intranuclear compartments. In addition, coordinately regulated astrocyte-specific genes on distinct chromosomes spatially associate in their inactive state and dissociate upon activation. These results provide direct evidence for function-related differential positioning of individual gene alleles within the interphase nucleus.[Keywords: Monoallelic expression; spatial gene positioning; nuclear architecture; astrocyte] Supplemental material is available at http://www.genesdev.org.

Highlights d Circadian depletion of GCs is required for reactivation of GR target genes d Ultradian and constant hormone treatments result in distinct RNA bursting patterns d The GR dwell time and bound fraction determine RNA burst duration and frequency d Stimulation with GCs induces co-bursting of proximal and distal transcription sites

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis-acting RNA element called the ‘packaging signal’ (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.DOI: http://dx.doi.org/10.7554/eLife.27055.001

No abstract

Automatic segmentation of cell nuclei is critical in several high-throughput cytometry applications whereas manual segmentation is laborious and irreproducible. One such emerging application is measuring the spatial organization (radial and relative distances) of fluorescence in situ hybridization (FISH) DNA sequences, where recent investigations strongly suggest a correlation between nonrandom arrangement of genes to carcinogenesis. Current automatic segmentation methods have varying performance in the presence of nonuniform illumination and clustering, and boundary accuracy is seldom assessed, which makes them suboptimal for this application. The authors propose a modular and model-based algorithm for extracting individual nuclei. It uses multiscale edge reconstruction for contrast stretching and edge enhancement as well as a multiscale entropy-based thresholding for handling nonuniform intensity variations. Nuclei are initially oversegmented and then merged based on area followed by automatic multistage classification into single nuclei and clustered nuclei. Estimation of input parameters and training of the classifiers is automatic. The algorithm was tested on 4,181 lymphoblast nuclei with varying degree of background nonuniformity and clustering. It extracted 3,515 individual nuclei and identified single nuclei and individual nuclei in clusters with 99.8 AE 0.3% and 95.5 AE 5.1% accuracy, respectively. Segmented boundaries of the individual nuclei were accurate when compared with manual segmentation with an average RMS deviation of 0.26 lm ($2 pixels). The proposed segmentation method is efficient, robust, and accurate for segmenting individual nuclei from fluorescence images containing clustered and isolated nuclei. The algorithm allows complete automation and facilitates reproducible and unbiased spatial analysis of DNA sequences. Published 2008 Wiley-Liss, Inc. Key termshigh-throughput segmentation; multiscale edge representation; nonuniform illumination; multiscale thresholding; watershed; region merging; cluster analysis IN recent years, investigations on nuclear architecture (spatial organization of nuclear organelles into well-defined compartments) and nonrandom gene positioning show significant impact on protein expression and cell functions (1-4). In these studies, the investigators used 2-D spatial distributions of relative and radial distances of fluorescence in situ hybridization (FISH)-labeled DNA sequences in interphase nuclei. They established the correlation between the spatial proximity of translocation prone genes and carcinogenesis (5-7). The motivation for this article stems from this interesting application in genomic organization. Our ultimate goal is to develop an exploratory data analysis system for studying the correlation between genomic organization and carcinogenesis (8). Naturally, such a system requires a segmentation method that can efficiently extract nuclei from fluorescence images and also possesses a high degree of segmentation accuracy (inaccurate segmentation ca...

TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These preexisting microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters LAT and Grb2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could in part explain the rapid kinetics with which TCR signal transduction occurs.