Biology subjects

Cremer, T.

Publications and source records attributed to Cremer, T..

7 recordsLinked to original sources

bioRxiv2024

Space-time dynamics of genome replication studied with super-resolved microscopy

Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.

cell biology↗

bioRxiv2023

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic {beta}-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic {beta}-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of {beta}-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation. These results indicate that insulin-derived peptides, presented by HLA-class I molecules at the cell surface, originate from ER-resident proinsulin that has been dislocated to the cytosol for subsequent degradation. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future therapies that may cure or even prevent T1D. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=172 HEIGHT=200 SRC="FIGDIR/small/545333v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@131d5org.highwire.dtl.DTLVardef@6eb4b3org.highwire.dtl.DTLVardef@196674org.highwire.dtl.DTLVardef@1957af6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Vimentin intermediate filaments organize organellar architecture in response to ER stress

Compartmentalization of organelles in space and time affects their functional state and enables higher order regulation of essential cellular processes. How organellar residence is maintained in a defined area of the cell remains poorly understood. In this study, we uncover a new role for intermediate filaments in the maintenance of organellar architecture and dynamics, which is executed through a functional connection between Vimentin and the ER-embedded ubiquitin ligase ring finger protein 26 (RNF26). While the ubiquitin ligase function of RNF26 promotes perinuclear positioning of endolysosomes, its catalytically inactive mutant I382R preferentially binds Vimentin through the RNF26 C-terminal tail. Loss of either RNF26 or Vimentin redistributes endolysosomes throughout the cytosol and mobilizes ER membranes from the perinuclear ER towards the periphery. Furthermore, RNF26 and Vimentin control changes in ER morphology and organelle compartmentalization during ER stress. Collectively, we define a new function for Vimentin-containing intermediate filaments as anchors of a dynamic interplay between the ER and endosomes, critical to the integrity of the perinuclear ER and corresponding perinuclear endosomal cloud during homeostatic and stress conditions. SynopsisThe perinuclear area hosts a wide variety of cellular organelles, and their interaction with the ER governs essential cellular processes. To spatiotemporally organize endosomes and ER in the perinuclear region, the ER-embedded E3 ubiquitin ligase RNF26 interacts with Vimentin to physically link the perinuclear ER membrane with the intermediate filament cytoskeleton. As a result, Vimentin ensures perinuclear RNF26 retention, which in turn controls the perinuclear location of ER membranes and endosomes, which can be affected during stressed conditions. O_LIVimentin interacts with inactive RNF26 in the ER membrane C_LIO_LIRNF26 by virtue of the Vimentin interaction controls perinuclear organization of ER membranes and the endosomal system C_LIO_LIVimentin immobilizes ER membranes in the perinuclear area C_LIO_LIVimentin and RNF26 compartmentalize organelles in the perinuclear region during ER stress C_LIO_LIWe define a new function of Vimentin intermediate filaments in the control of the perinuclear endosomal and ER organization C_LI

cell biology↗

bioRxiv2022

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggested only modest compaction differences ([~]2-10x) between active and inactive nuclear compartments (ANC and INC). Here, we present maps of nuclear landscapes with true-to-scale DNA-densities, ranging from <5 Mbp/{micro}m3 to >300 Mbp/{micro}m3. Maps were generated from individual human and mouse cell nuclei with single-molecule localization microscopy at [~]20 nm lateral and [~]100 nm axial resolution and supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription and replication into nuclei of living cells, demonstrated their localization and movements within the ANC and exclusion from the INC.

cell biology↗

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗

bioRxiv2020

ER-embedded UBE2J1/RNF26 ubiquitylation complex in spatio-temporal control of the endolysosomal pathway

The endolysosomal system fulfills a wide variety of cellular functions, many of which are modulated through interactions with other organelles. In particular, the ER exerts spatiotemporal constraints on the organization and motility of endosomes and lysosomes. We have recently described the ER transmembrane E3 ubiquitin ligase RNF26 to control perinuclear positioning and transport dynamics of the endolysosomal vesicular network. We now report that the ubiquitin conjugating enzyme UBE2J1, also anchored in the ER membrane, collaborates with RNF26 in this context, and that the cellular activity of this E2/E3 pair, localized in a perinuclear ER subdomain, is underpinned by transmembrane interactions. Through modification of its substrate SQSTM1/p62, the ER-embedded UBE2J1/RNF26 ubiquitylation complex recruits endosomal adaptors to immobilize their cognate vesicles in the perinuclear region. The resulting spatiotemporal compartmentalization of the endolysosomal system between the perinuclear vesicle cloud and the cell periphery facilitates timely downregulation of endocytosed cargoes, such as EGFR.

cell biology↗

bioRxiv2019

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is debatable. Using live-cell and super-resolved 3D microscopy, we demonstrate that cohesin depleted cells pass through an endomitosis and rebuild a single multilobulated nucleus (MLN) with chromosome territories (CTs) pervaded by interchromatin channels. CTs contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. Splicing speckles are located nearby within the lining channel system. These clusters form microscopically defined, active and inactive compartments, which correspond to A/B compartments, detected with ensemble Hi-C. Functionality of MLN despite continuous absence of cohesin was demonstrated by their ability to 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.

cell biology↗