PubMed Health⌕ Search

Biomedical subjects

Lothar Schermelleh

Publications and source records attributed to Lothar Schermelleh.

11 recordsLinked to original sources
PubMed2026

Cohesin prevents local mixing of condensed euchromatic domains in living human cells.

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, how cohesin organizes these domains in living cells, especially in active euchromatin, remains elusive. Here, to address this question, we combined single-nucleosome imaging/tracking and super-resolution three-dimensional structured illumination microscopy with euchromatin-specific labeling of histone variant H3.3. Using this nanoscopic approach, we revealed that euchromatin forms condensed domains that are constrained by cohesin-mediated loops. This organization refines the classical view of euchromatin as largely open, in line with emerging evidence. Transcription machinery appears to be located near the condensed domain surfaces/borders. Cohesin loss increased nucleosome-level fluidity within these domains without altering their overall compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings suggest a physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

Humans↗
PubMed2006

Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks.

In spite of strong evidence that the nucleus is a highly organized organelle, a consensus on basic principles of the global nuclear architecture has not so far been achieved. The chromosome territory-interchromatin compartment (CT-IC) model postulates an IC which expands between chromatin domains both in the interior and the periphery of CT. Other models, however, dispute the existence of the IC and claim that numerous chromatin loops expand between and within CTs. The present study was undertaken to resolve these conflicting views. (1) We demonstrate that most chromatin exists in the form of higher-order chromatin domains with a compaction level at least 10 times above the level of extended 30 nm chromatin fibers. A similar compaction level was obtained in a detailed analysis of a particularly gene-dense chromosome region on HSA 11, which often expanded from its CT as a finger-like chromatin protrusion. (2) We further applied an approach which allows the experimental manipulation of both chromatin condensation and the width of IC channels in a fully reversible manner. These experiments, together with electron microscopic observations, demonstrate the existence of the IC as a dynamic, structurally distinct nuclear compartment, which is functionally linked with the chromatin compartment.

Animals↗
PubMed2006

Targeting and tracing antigens in live cells with fluorescent nanobodies.

We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

Animals↗
PubMed2005

Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming.

BACKGROUND: Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage. RESULTS: Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos. CONCLUSION: The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantation development.

Animals↗
PubMed2005

Recruitment of DNA methyltransferase I to DNA repair sites.

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

Animals↗
PubMed2005

Trapped in action: direct visualization of DNA methyltransferase activity in living cells.

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.

Animals↗
PubMed2004

Differences in centromere positioning of cycling and postmitotic human cell types.

Centromere positioning in human cell nuclei was traced in non-cycling peripheral blood lymphocytes (G0) and in terminally differentiated monocytes, as well as in cycling phytohemagglutinin-stimulated lymphocytes, diploid lymphoblastoid cells, normal fibroblasts, and neuroblastoma SH-EP cells using immunostaining of kinetochores, confocal microscopy and three-dimensional image analysis. Cell cycle stages were identified for each individual cell by a combination of replication labeling with 5-bromo-2'-deoxyuridine and immunostaining of pKi67. We demonstrate that the behavior of centromeres is similar in all cell types studied: a large fraction of centromeres are in the nuclear interior during early G1; in late G1 and early S phase, centromeres shift to the nuclear periphery and fuse in clusters. Peripheral location and clustering of centromeres are most pronounced in non-cycling cells (G0) and terminally differentiated monocytes. In late S and G2, centromeres partially decluster and migrate towards the nuclear interior. In the rather flat nuclei of adherently growing fibroblasts and neuroblastoma cells, kinetochores showed asymmetrical distributions with preferential kinetochore location close either to the bottom side of the nucleus (adjacent to the growth surface) or to the nuclear upper side. This asymmetrical distribution of centromeres is considered to be a consequence of chromosome arrangement in anaphase rosettes.

Bromodeoxyuridine↗
PubMed2004

Replication-independent chromatin loading of Dnmt1 during G2 and M phases.

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation.

Animals↗
PubMed2003

Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages.

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several microm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of approximately 1 microm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.

Cell Movement↗
PubMed2002

Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH).

3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH procedure. For in vivo studies we employed replication labeling of DNA with Cy3-conjugated nucleotides and/or chromatin labeling by GFP-tagged histone 2B. At the light microscope level, we compared the intranuclear distribution of H2B-GFP-tagged chromatin and the positions of replication-labeled chromatin domains in the same individual cells in vivo, after fixation with 4% paraformaldehyde, and after 3D-FISH. Light microscope data demonstrate a high degree of preservation of the spatial arrangement of approximately 1-Mb chromatin domains. Subsequent electron microscope investigations of chromatin structure showed strong alterations in the ultrastructure of the nucleus caused mainly by the heat denaturation step. Through this step chromatin acquires the appearance of a net with mesh size of 50-200 nm roughly corresponding to the average displacement of the chromatin domains observed at light microscope level. We conclude that 3D-FISH is a useful tool to study chromosome territory structure and arrangements down to the level of approximately 1-Mb chromatin domain positions. However, important ultrastructural details of the chromatin architecture are destroyed by the heat denaturation step, thus putting a limit to the usefulness of 3D-FISH analyses at nanometer scales.

Carbocyanines↗