Nucleus (Austin, Tex.)最新文献

Activation of transcription results in coordinated movement of chromatin over a range of micrometers. To investigate how transcriptional regulation affects the mobility of RNA Pol II molecules and whether this movement response depends on the coordinated movement of chromatin, we used our Dense Flow reConstruction and Correlation (DFCC) method. Using DFCC, we studies the nucleus-wide coherent movements of RNA Pol II in the context of DNA in humancancer cells. This study showed the dependance of coherent movements of RNA Pol II molecules (above 1 µm) on transcriptional activity. Here, we share the dataset of this study, includes nucleus-wide live imaging and analysis of DNA and RNA polymerase II in different transcription states, and the code for teh analysis. Our dataset may provide researchers interested in the long-range organization of chromatin in living cell images with the ability to link the structural genomic compartment to dynamic information. .

There is growing appreciation that architectural components of the nucleus regulate gene accessibility by altering chromatin organization. While nuclear membrane connector proteins link the mechanosensitive actin cytoskeleton to the nucleoskeleton, actin's contribution to the inner architecture of the nucleus remains enigmatic. Control of actin transport into the nucleus, plus the presence of proteins that control actin structure (the actin tool-box) within the nucleus, suggests that nuclear actin may support biomechanical regulation of gene expression. Cellular actin structure is mechanoresponsive: actin cables generated through forces experienced at the plasma membrane transmit force into the nucleus. We posit that dynamic actin remodeling in response to such biomechanical cues provides a novel level of structural control over the epigenetic landscape. We here propose to bring awareness to the fact that mechanical forces can promote actin transfer into the nucleus and control structural arrangements as illustrated in mesenchymal stem cells, thereby modulating lineage commitment.

The kinetochore is a large proteinaceous structure assembled on the centromeres of chromosomes. The complex machinery links chromosomes to the mitotic spindle and is essential for accurate chromosome segregation during cell division. The kinetochore is composed of two submodules: the inner and outer kinetochore. The inner kinetochore is assembled on centromeric chromatin and persists with centromeres throughout the cell cycle. The outer kinetochore attaches microtubules to the inner kinetochore, and assembles only during mitosis. The review focuses on recent advances in our understanding of the mechanisms governing the proper assembly of the outer kinetochore during mitosis and highlights open questions for future investigation.

Access to DNA is a prerequisite to the execution of essential cellular processes that include transcription, replication, chromosomal segregation, and DNA repair. How the proteins that regulate these processes function in the context of chromatin and its dynamic architectures is an intensive field of study. Over the past decade, genome-wide assays and new imaging approaches have enabled a greater understanding of how access to the genome is regulated by nucleosomes and associated proteins. Additional mechanisms that may control DNA accessibility in vivo include chromatin compaction and phase separation - processes that are beginning to be understood. Here, we review the ongoing development of accessibility measurements, we summarize the different molecular and structural mechanisms that shape the accessibility landscape, and we detail the many important biological functions that are linked to chromatin accessibility.

The Nuclear Pore Complex (NPC) represents a critical passage through the nuclear envelope for nuclear import and export that impacts nearly every cellular process at some level. Recent technological advances in the form of Auxin Inducible Degron (AID) strategies and Single-Point Edge-Excitation sub-Diffraction (SPEED) microscopy have enabled us to provide new insight into the distinct functions and roles of nuclear basket nucleoporins (Nups) upon nuclear docking and export for mRNAs. In this paper, we provide a review of our recent findings as well as an assessment of new techniques, updated models, and future perspectives in the studies of mRNA's nuclear export.

Heterochromatin is the highly compacted form of chromatin with various condensation levels hallmarked by high DNA methylation. MeCP2 is mostly known as a DNA methylation reader but has also been reported as a heterochromatin organizer. Here, we combine liquid-liquid phase separation (LLPS) analysis and single-molecule tracking with quantification of local MeCP2 concentrations in vitro and in vivo to explore the mechanism of MeCP2-driven heterochromatin organization and dynamics. We show that MeCP2 alone forms liquid-like spherical droplets via multivalent electrostatic interactions and with isotropic mobility. Crowded environments and DNA promote MeCP2 LLPS and slow down MeCP2 mobility. DNA methylation, however, restricts the growth of heterochromatin compartments correlating with immobilization of MeCP2. Furthermore, MeCP2 self-interaction is required for LLPS and is disrupted by Rett syndrome mutations. In summary, we are able to model the heterochromatin compartmentalization as well as MeCP2 concentration and heterogeneous motion in the minimal in vitro system.

Nuclear lamins and transport are intrinsically linked, but their relationship is yet to be fully unraveled. A multitude of complex, coupled interactions between lamins and nucleoporins (Nups), which mediate active transport into and out of the nucleus, combined with well documented dysregulation of lamins in many cancers, suggests that lamins and nuclear transport may play a pivotal role in carcinogenesis and the preservation of cancer. Changes of function related to lamin/Nup activity can principally lead to DNA damage, further increasing the genetic diversity within a tumor, which could lead to the reduction the effectiveness of antineoplastic treatments. This review discusses and synthesizes different connections of lamins to nuclear transport and offers a number of outlook questions, the answers to which could reveal a new perspective on the connection of lamins to molecular transport of cancer therapeutics, in addition to their established role in nuclear mechanics.

Autophagy has emerged as a key regulator of cell metabolism. Recently, we have demonstrated that autophagy is involved in RNA metabolism by regulating ribosomal RNA (rRNA) synthesis. We found that autophagy-deficient cells display much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling, which facilitates the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoter regions and leads to the activation of rDNA transcription. Finally, we showed that SQSTM1 accumulation is responsible for the increase in protein synthesis, cell growth and cell proliferation in autophagy-deficient cells. Taken together, our findings reveal a regulatory role of autophagy and autophagy receptor SQSTM1 in rRNA synthesis and may provide novel mechanisms for the hyperactivated rDNA transcription in autophagy-related human diseases.Abbreviations: 5-FUrd: 5-fluorouridine; LAP: MAP1LC3/LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A: RNA polymerase I subunit A; rDNA: ribosomal DNA; RRN3: RRN3 homolog, RNA polymerase I transcription factor; rRNA: ribosomal RNA; SQSTM1/p62: sequestosome 1; TP53INP2: tumor protein p53 inducible nuclear protein 2; UBTF: upstream binding transcription factor.

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.