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#33091376   2020/10/19 To Up

The relative densities of cytoplasm and nuclear compartments are robust against strong perturbation.

The cell nucleus is a compartment in which essential processes such as gene transcription and DNA replication occur. While the large amount of chromatin confined in the finite nuclear space could install the picture of a particularly dense organelle surrounded by less dense cytoplasm, recent studies have begun to report the opposite. However, the generality of this newly emerging, opposite picture has so far not been tested. Here, we used combined optical diffraction tomography (ODT) and epi-fluorescence microscopy to systematically quantify the mass densities of cytoplasm, nucleoplasm, and nucleoli of human cell lines, challenged by various perturbations. We found that the nucleoplasm maintains a lower mass density than cytoplasm during cell cycle progression by scaling its volume to match the increase of dry mass during cell growth. At the same time, nucleoli exhibited a significantly higher mass density than the cytoplasm. Moreover, actin and microtubule depolymerization and changing chromatin condensation altered volume, shape, and dry mass of those compartments, while the relative distribution of mass densities was generally unchanged. Our findings suggest that the relative mass densities across membrane-bound and membraneless compartments are robustly conserved, likely by different as of yet unknown mechanisms, which hints at an underlying functional relevance. This surprising robustness of mass densities contributes to an increasing recognition of the importance of physico-chemical properties in determining cellular characteristics and compartments.
Kyoohyun Kim, Jochen Guck

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#33091336   2020/10/14 To Up

Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging.

CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.
Bo Gu, Colin J Comerci, Dannielle G McCarthy, Saumya Saurabh, W E Moerner, Joanna Wysocka

2397 related Products with: Opposing Effects of Cohesin and Transcription on CTCF Organization Revealed by Super-resolution Imaging.

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#33091009   2020/10/22 To Up

Effect of H2A.Z deletion is rescued by compensatory mutations in Fusarium graminearum.

Fusarium head blight is a destructive disease of grains resulting in reduced yields and contamination of grains with mycotoxins worldwide; Fusarium graminearum is its major causal agent. Chromatin structure changes play key roles in regulating mycotoxin biosynthesis in filamentous fungi. Using a split-marker approach in three F. graminearum strains INRA156, INRA349 and INRA812 (PH-1), we knocked out the gene encoding H2A.Z, a ubiquitous histone variant reported to be involved in a diverse range of biological processes in yeast, plants and animals, but rarely studied in filamentous fungi. All ΔH2A.Z mutants exhibit defects in development including radial growth, sporulation, germination and sexual reproduction, but with varying degrees of severity between them. Heterogeneity of osmotic and oxidative stress response as well as mycotoxin production was observed in ΔH2A.Z strains. Adding-back wild-type H2A.Z in INRA349ΔH2A.Z could not rescue the phenotypes. Whole genome sequencing revealed that, although H2A.Z has been removed from the genome and the deletion cassette is inserted at H2A.Z locus only, mutations occur at other loci in each mutant regardless of the genetic background. Genes affected by these mutations encode proteins involved in chromatin remodeling, such as the helicase Swr1p or an essential subunit of the histone deacetylase Rpd3S, and one protein of unknown function. These observations suggest that H2A.Z and the genes affected by such mutations are part or the same genetic interaction network. Our results underline the genetic plasticity of F. graminearum facing detrimental gene perturbation. These findings suggest that intergenic suppressions rescue deleterious phenotypes in ΔH2A.Z strains, and that H2A.Z may be essential in F. graminearum. This assumption is further supported by the fact that H2A.Z deletion failed in another Fusarium spp., i.e., the rice pathogen Fusarium fujikuroi.
Zhenhui Chen, Enric Zehraoui, Anna K Atanasoff-Kardjalieff, Joseph Strauss, Lena Studt, Nadia Ponts

1933 related Products with: Effect of H2A.Z deletion is rescued by compensatory mutations in Fusarium graminearum.

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#33090576   2020/10/08 To Up

Molecular pathogenesis of cutaneous lymphoma -future directions.

The pathogenesis of cutaneous T-cell lymphomas is not clear. In recent years the genetic changes in CTCL were explored. The detected mutations showed a great deal of heterogeneity between individual patients. The studies documented various copy number variations (CNV) and single nucleotide variations (SNV) in multiple genes involved in multiple signaling pathways. Recurrently mutated signaling pathways include JAK-STAT, MAPK, T cell receptor, TNF receptor and NFκB signaling. In the period between 2018 and today additional studies towards the genetic changes in CTCL were carried out. Genetic changes in gamma delta T cell lymphoma are also shown in genes of the JAK-STAT, MAPK, MYC and chromatin signaling pathways. These studies might indicate a shift away from targeted sequencing approaches towards whole genome sequencing. This approach demands additional resources in terms of funding but has the advantage of finding mutations in non-coding regions. These mutations were neglected for a long time, but as shown in contemporary research these regions harbor highly recurrent mutations affecting gene expression and regulation. Nevertheless, the detection of specific molecular changes in known pathways enables considerations for targeted therapies.
Rudolf Stadler, Carsten Hain, Cassandra Cieslak, René Stranzenbach

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#33089509   2020/10/22 To Up

Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation.

Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.
Stefan Biedzinski, Gökçe Agsu, Benoit Vianay, Marc Delord, Laurent Blanchoin, Jerome Larghero, Lionel Faivre, Manuel Théry, Stéphane Brunet

1637 related Products with: Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation.

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