Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism.This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs. INTRODUCTIONTelomeres are specialized chromatin structures at the end of linear chromosomes, in which repetitive DNA sequences (5Ј-TTAGGG-3Ј in vertebrates) associate into a nucleoprotein complex (de Lange et al., 2006). This complex-the "telosome"-protects the chromosome ends from degradation and genomic rearrangement and includes the "shelterin" proteins TRF1, TRF2, and POT1 that directly recognize the TTAGGG repeat sequence (de Lange, 2005;Bertuch and Lundblad, 2006;Croy and Wuttke, 2006;Blasco, 2007). Because of incomplete DNA synthesis at the chromosome ends, 50 -200 base pairs of telomeric DNA are lost during each replication cycle (Harley et al., 1990;Martens et al., 2000). After ϳ60 -80 cell divisions telomere repeats are shortened from a typical initial length of 10 -15 kb in human cells to ϳ5 kb and below, which triggers cell senescence or apoptosis (Harley et al., 1990;Martens et al., 2000;Blasco, 2007). Accordingly, tumor cells need to compensate the loss of their telomere repeats in order to sustain an unlimited proliferative potential. In most cases this is accomplished by reactivating telomerase, a reverse transcriptase that synthesizes telomeric repeats at the chromosome ends (Greider and Blackburn, 1985;Chan and Blackburn, 2004;Collado et al., 2007;Johnson and Broccoli, 2007). However, a fraction of ϳ10 -15% of tumors is able to maintain their telomeres in the absence of telomerase activity. This process has been designated as alternative lengthening of telomeres (ALT; Bryan et al., 1995;Neumann and Reddel, 2006;Johnson and Broccoli, 2007). In yeast and mammals, it has been shown that the ALT mechanism involves homologous recombination events between telomere repeats (Dunham et al., 2000;Kass-Eisler and Greider, 2000;Lundblad, 2002;Muntoni and Reddel, 2005). It is characte...

SummaryPromyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50-to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PMLNBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

SummaryThe position of the nucleus is regulated in different developmental stages and cellular events. During polarization, the nucleus moves away from the future leading edge and this movement is required for proper cell migration. Nuclear movement requires the LINC complex components nesprin-2G and SUN2, which form transmembrane actin-associated nuclear (TAN) lines at the nuclear envelope. Here we show that the nuclear envelope protein Samp1 (NET5) is involved in nuclear movement during fibroblast polarization and migration. Moreover, we demonstrate that Samp1 is a component of TAN lines that contain nesprin-2G and SUN2. Finally, Samp1 associates with SUN2 and lamin A/C, and the presence of Samp1 at the nuclear envelope requires lamin A/C. These results support a role for Samp1 in the association between the LINC complex and lamins during nuclear movement.

Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil-and waterimmersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution. Abbreviations

Telomere positioning near the nuclear envelope during Caenorhabditis elegans development requires the nuclear envelope protein SUN-1, the Shelterin component POT-1, and the SUMO ligase GEI-17.

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