Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in ...great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate ...super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.
Lipid droplets (LDs) are ubiquitous organelles comprising a central hub for cellular lipid metabolism and trafficking. This role is tightly associated with their interactions with several cellular ...organelles. Here, we provide a systematic and quantitative structural description of LDs in their native state in HeLa cells enabled by cellular cryoelectron microscopy. LDs consist of a hydrophobic neutral lipid mixture of triacylglycerols (TAG) and cholesteryl esters (CE), surrounded by a single monolayer of phospholipids. We show that under normal culture conditions, LDs are amorphous and that they transition into a smectic liquid-crystalline phase surrounding an amorphous core at physiological temperature under certain cell-cycle stages or metabolic scenarios. Following determination of the crystal lattice spacing of 3.5 nm and of a phase transition temperature below 43 °C, we attributed the liquid-crystalline phase to CE. We suggest that under mitotic arrest and starvation, relative CE levels increase, presumably due to the consumption of TAG metabolites for membrane synthesis and mitochondrial respiration, respectively, supported by direct visualization of LD–mitochondrial membrane contact sites. We hypothesize that the structural phase transition may have a major impact on the accessibility of lipids in LDs to enzymes or lipid transporters. These may become restricted in the smectic phase, affecting the exchange rate of lipids with surrounding membranes and lead to a different surface occupancy of LD-associated proteins. Therefore, the composition and the resulting internal structure of LDs is expected to play a key role in their function as hubs of cellular lipid flux.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and ...super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the ...replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.
RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible ...liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.
Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic ...regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4-20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as "chromocenter scattering." We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4-20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation.
Intestinal epithelial barrier properties are maintained by a junctional complex consisting of tight junctions (TJ), adherens junctions (AJ) and desmosomes. Desmoglein 2 (Dsg2), an adhesion molecule ...of desmosomes and the only Dsg isoform expressed in enterocytes, is required for epithelial barrier properties and may contribute to barrier defects in Crohn's disease. Here, we identified extradesmosomal Dsg2 on the surface of polarized enterocytes by Triton extraction, confocal microscopy, SIM and STED. Atomic force microscopy (AFM) revealed Dsg2-specific binding events along the cell border on the surface of enterocytes with a mean unbinding force of around 30pN. Binding events were blocked by an inhibitory antibody targeting Dsg2 which under same conditions activated p38MAPK but did not reduce cell cohesion. In enterocytes deficient for Dsg2, p38MAPK activity was reduced and both barrier integrity and reformation were impaired. Dsc2 rescue did not restore p38MAPK activity indicating that Dsg2 is required. Accordingly, direct activation of p38MAPK in Dsg2-deficient cells enhanced barrier reformation demonstrating that Dsg2-mediated activation of p38MAPK is crucial for barrier function. Collectively, our data show that Dsg2, beside its adhesion function, regulates intestinal barrier function via p38MAPK signalling. This is in contrast to keratinocytes and points towards tissue-specific signalling functions of desmosomal cadherins.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities ...have been identified including, among others, demethylation of
-methylcytosine (m
C) in RNA- and single-stranded DNA oligonucleotides, demethylation of
-methyladenosine (m
A) in tRNA or formation of 5-formyl cytosine (f
C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in
, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPR
) in
.
Sorting and export of transmembrane cargoes and lysosomal hydrolases at the trans-Golgi network (TGN) are well understood. However, elucidation of the mechanism by which secretory cargoes are ...segregated for their release into the extracellular space remains a challenge. We have previously demonstrated that, in a reaction that requires Ca(2+), the soluble TGN-resident protein Cab45 is necessary for the sorting of secretory cargoes at the TGN. Here, we report that Cab45 reversibly assembles into oligomers in the presence of Ca(2+) These Cab45 oligomers specifically bind secretory proteins, such as COMP and LyzC, in a Ca(2+)-dependent manner in vitro. In intact cells, mutation of the Ca(2+)-binding sites in Cab45 impairs oligomerization, as well as COMP and LyzC sorting. Superresolution microscopy revealed that Cab45 colocalizes with secretory proteins and the TGN Ca(2+) pump (SPCA1) in specific TGN microdomains. These findings reveal that Ca(2+)-dependent changes in Cab45 mediate sorting of specific cargo molecules at the TGN.
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