Biology subjects

Wagner, T.

Publications and source records attributed to Wagner, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

In situ structures from relaxed cardiac myofibrils reveal the organization of the muscle thick filament

The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit of striated muscle1. Mutations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2, 3. Despite this central importance for sarcomere force generation, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction. Here, we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by electron cryo-tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin heads and tails, myosin-binding protein C (MyBP-C) and titin, elucidating the structural basis for their interaction during muscle contraction. The arrangement of myosin heads is variable depending on their position along the filament, suggesting that they have different capacities in terms of strain susceptibility and activation. Myosin tails exhibit a distinct arrangement and pattern of interactions. These are likely orchestrated by three alpha and three beta titin chains that are arranged like a spring, suggesting the existence of specialized roles of thick filament segments in length-dependent activation and contraction. Surprisingly, while the three titin alpha chains run along the entire length of the thick filament, titin beta does not. The structure also demonstrates that the C-terminal region of MyBP-C binds myosin tails and unexpectedly also directly interacts with the myosin heads, suggesting a previously undescribed direct role in the preservation of the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. These findings establish a robust groundwork for forthcoming research endeavors aiming to explore muscle disorders that involve sarcomeric structural components.

biochemistry↗

bioRxiv2023

Differences in the regulation mechanisms of the glutamine synthetase from methanogenic archaea unveiled by structural investigations

Glutamine synthetases catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be tightly regulated to fit cellular needs. While their catalytic mechanisms and regulation are well-characterized in bacteria and eukaryotes, only limited knowledge exists about the archaeal representatives. Here, we natively purified the glutamine synthetases type I- from Methanothermococcus thermolithotrophicus and Methermicoccus shengliensis, two thermophilic methanogens belonging to different orders. Biochemical investigations combined with X-ray crystallography unveiled the first structures of archaeal glutamine synthetases and highlighted differences in their regulation. The enzyme from M. thermolithotrophicus is inactive in its resting state and employs 2-oxoglutarate as an on-switch. The 2-oxoglutarate acts as a sensor of cellular nitrogen deficiency, and its reported cellular concentration remarkably overlays with that required for the enzyme activation. Its binding to an allosteric pocket leads to the reconfiguration of the active site and promotes a catalytically competent state. The homolog from M. shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited by glutamine, as shown for bacterial homologs. The glutamine inhibition depends on a key arginine residue from the Asp50-loop. The arginine is substituted by a glycine in M. thermolithotrophicus, abolishing the inhibitory effect. While the effectors are surprisingly different, the molecular switch controlling the glutamine synthetase activity is fundamentally the same and depends on the correct positioning of the Asp50-loop and a catalytic arginine. Residue conservation suggests that both regulation mechanisms are widespread and not mutually exclusive across archaea.

biochemistry↗

bioRxiv2023

High resolution cryo-EM and crystallographic snapshots of the large actinobacterial 2-oxoglutarate dehydrogenase: an all-in-one fusion with unique properties

Actinobacteria possess unique ways to regulate the oxoglutarate node located in the central position of the tricarboxylic acid cycle, a crossroad between energy conservation and nitrogen metabolism. Here, we studied the decarboxylative oxidation route that leads, through the 2-oxoglutarate dehydrogenase (ODH) complex, to the generation of succinyl-CoA and reduced equivalents to feed the respiratory chain. Compared to most organisms in which the oxidative decarboxylation and reductive acylation steps are carried out by different enzymes within the ODH complex, actinobacteria rely on an all-in-one protein (OdhA) in which both activities are carried out by the same polypeptide. We describe high-resolution cryo-EM and X-ray crystallography snapshots of representative enzymes from Mycobacterium smegmatis and Corynebacterium glutamicum, showing that OdhA is an 800-kDa homohexamer that folds into a three-blade propeller shape. The obligate trimeric and dimeric states of the acyltransferase and dehydrogenase domains, respectively, are critical for maintaining the overall assembly, where both domains interact via subtle readjustments of their interfaces. Complexes obtained with substrate analogues, reaction products and allosteric regulators illustrate how these domains operate. Furthermore, we provide additional insights into the phosphorylation-dependent regulation of this enzymatic machinery by the FHA (Fork-Head Associated) signalling protein OdhI, delivering new molecular details on how this actinobacterial-specific switching mechanism operates. Overall, the quaternary organization of OdhA represents a new piece of the fascinating puzzle of the synergistic, mixed pyruvate dehydrogenase/2-oxoglutarate dehydrogenase actinobacterial supercomplex.

biochemistry↗

bioRxiv2023

Specialized pathogenic cells release Tc toxins using a type 10 secretion system

Disease-causing bacteria use a variety of secreted toxins to invade and subjugate their hosts. While the machinery responsible for secretion of many smaller toxins has already been established, it remains enigmatic for larger ones such as Tc toxins from human and insect pathogens, which approach the size of a prokaryotic ribosome. In the present study, we combine targeted genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga to reveal that a specialized subset of bacterial cells produces the Tc toxin YenTc as part of a complex toxin cocktail released into the environment by controlled cell lysis using a transcriptionally-coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the process of Tc toxin export by a T10SS in hitherto unprecedented detail, identifying that T10SSs operate via a previously unknown lytic mode of action, and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major human pathogens such as Yersinia pestis and Salmonella enterica, we anticipate our findings to model an important aspect of pathogenesis in bacteria with a significant impact on global human health.

microbiology↗

bioRxiv2022

How a methanogen assimilates sulfate: Structural and functional elucidation of the complete sulfate-reduction pathway

By growing on sulfate as the sole source of sulfur, Methanothermococcus thermolithotrophicus breaks a dogma: the ancient metabolic pathways methanogenesis and sulfate-reduction should not co-occur in one organism due to toxic intermediates and energetic barriers. Using a complementary approach of physiological, biochemical, and structural studies, we provide a snapshot of the complete sulfate-reduction pathway of the methanogenic archaeon. While the first two reactions proceed via an ATP-sulfurylase and APS-kinase, common to other organisms, the further steps are catalysed by non-canonical enzymes. 3-phosphoadenosine-5-phosphosulfate (PAPS) released by the APS-kinase is converted into sulfite and 3-phosphoadenosine-5-phosphate (PAP) by a new class of PAPS-reductase that shares high similarity with the APS-reductases involved in dissimilatory sulfate-reduction. The generated PAP is efficiently hydrolysed by a PAP-phosphatase that was likely derived from an RNA exonuclease. Finally, the F420-dependent sulfite-reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that genes of the sulfate-reduction pathway are present in various methanogens, M. thermolithotrophicus uses a distinct way to assimilate sulfate. We propose that its entire sulfate-assimilation pathway was derived from a "mix-and-match" strategy in which the methanogen acquired assimilatory and dissimilatory enzymes from other microorganisms and shaped them to fit its physiological needs.

microbiology↗

bioRxiv2022

Transcriptomics sheds light on N2-fixation strategies employed by a thermophilic member of the Methanococcales

Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales, that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadate. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery including chaperones, regulators, and molybdate-importers, as well as simultaneous upregulation of an ammonium-transporter and a putative pathway for nitrate/nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. ImportanceThe thermophilic anaerobic archaeon, M. thermolithotrophicus, is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability to simultaneously reduce N2/CO2 into NH3/CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2-fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance by investigating its physiology and uncharacterized nitrogenase. We demonstrate that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.

microbiology↗

bioRxiv2022

TomoTwin: Generalized 3D Localization of Macromolecules in Cryo-electron Tomograms with Structural Data Mining

Cryoelectron tomography enables the visualization of cellular environments in extreme detail through the lens of a benign observer; what remains lacking however are tools to analyze the full amount of information contained within these densely packed volumes. Detailed analysis of macromolecules through subtomogram averaging requires particles to first be localized within the tomogram volume, a task complicated by several factors including a low signal to noise ratio and crowding of the cellular space. Available methods for this task suffer either from being error prone or requiring manual annotation of training data. To assist in this crucial particle picking step, we present TomoTwin: a robust, first in class general picking model for cryo-electron tomograms based on deep metric learning. By embedding tomograms in an information-rich, high-dimensional space which separates macromolecules according to their 3-dimensional structure, TomoTwin allows users to identify proteins in tomograms de novo without manually creating training data or retraining the network each time a new protein is to be located. TomoTwin is open source and available at https://github.com/MPI-Dortmund/tomotwin-cryoet.

biophysics↗

bioRxiv2022

A systematic examination of learning in the invasive ant Linepithema humile reveals very rapid development of short and long-term memories

The Argentine ant (Linepithema humile) is one of the most damaging and widespread invasive ant species worldwide. However, control attempts often fail due to bait rejection or insufficient bait uptake. Increasing preference for, and consumption of, bait is thus an important requirement for successful control. Learning and within-nest information transfer might be a potential tool for achieving this goal. We conducted a systematic investigation of olfactory learning and route learning in Argentine ants. The ants showed very strong and rapid route learning, choosing the correct arm in a Y-maze 65% of time after just one visit, and 84% correct after two. Odour learning was even more rapid, reaching up to 85% correct choices after just one exposure to flavoured food. Learning is long-lasting, with 73% correct choices after 48h. Food flavour information is transferred efficiently between nestmates in the nest, driving preference: naive ants housed with ants fed on flavoured food show a strong preference (77%) for that odour after 24h. Overall, Linepithema humile are outstanding learners. This, coupled with efficient intranidal information transfer and strong pheromonal recruitment, may help explain their ability to discover and then dominate resources. However, these strengths could potentially be used against them, by exploiting learning and information transfer to increase toxic bait uptake. Steering ant preference by leveraging learning might be an underappreciated tool in invasive alien species control.

animal behavior and cognition↗

bioRxiv2022

The structure of the F420-dependent sulfite-detoxifying enzyme from Methanococcales reveals a prototypical sulfite-reductase with assimilatory traits.

The coenzyme F420-dependent sulfite reductase (Fsr group I) protects hydrogenotrophic methanogens, one of the main contributors in worldwide methane emission, from toxic sulfite. Fsr is a single peptide composed of a F420H2-oxidase and a novel class of sulfite reductase. Both catalytic domains have been proposed to be the ancestors of modern F420-oxido/reductases and dissimilatory/assimilatory sulfite reductases. Here, we describe the X-ray crystal structures of Fsr natively isolated from Methanocaldococcus jannaschii (MjFsr) and Methanothermococcus thermolithotrophicus (MtFsr), respectively refined to 2.30 [A] and 1.55 [A] resolution. In both organisms, Fsr oligomerizes as a 280-kDa homotetramer, where each siroheme-[4Fe-4S] is catalytically active, in contrast to dissimilatory homologues. The siroheme-[4Fe-4S], embedded in the sulfite reductase domain, is electronically connected to the flavin in the F420H2-oxidase domain by five [4Fe-4S]-clusters. EPR spectroscopy determined the redox potentials of these [4Fe-4S]2+/1+ clusters (-435 to -275 mV), through which electrons flow from FAD to the siroheme-[4Fe-4S]2+/1+ (siroheme, -114 mV; [4Fe-4S] -445 mV). The electron relay is mainly organized by two inserted ferredoxin modules, which stabilize the higher degree of oligomerization. While the F420H2-oxidase part is similar to the {beta}-subunit of F420-reducing hydrogenases, the sulfite reductase domain is structurally analogous to dissimilatory sulfite reductases, whereas its siroheme-[4Fe-4S] cofactor is bound in the same way as in assimilatory ones. Accordingly, the reaction of MtFsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide the first structural insights into this unique fusion, a snapshot of a primitive sulfite reductase that turns a poison into an elementary block of Life.

biochemistry↗

bioRxiv2021

Structure of the thin filament in native skeletal muscles reveals its interaction with nebulin and two distinct conformations of myosin

Nebulin is a major structural protein of skeletal sarcomeres and is essential for proper assembly and contraction of skeletal muscle1. It stabilises and regulates the length of thin filaments,2 but the structural mechanism remains nebulous. Using electron cryotomography and sub-tomogram averaging, we present the first structure of native nebulin bound to thin filaments within the A-band and I-band of intact sarcomeres. This in-situ reconstruction reveals unprecedented detail of interaction at pseudo-atomic resolution between nebulin and actin, providing the basis for understanding the structural and regulatory roles of nebulin. The position of nebulin on the thin filament indicates that there is no contact to tropomyosin or myosin, but an unexpected interaction with a troponin-T linker, possibly through two binding motifs on nebulin. In addition, our structure of myosin bound to the thin filaments reveals different conformations of the neck domain, both within the same sarcomere and when compared to purified structures, highlighting an inherent structural variability in muscle. We provide a complete description of cross-bridge formation on fully native, nebulin-containing thin filaments at near-atomic scale. Our structures establish the molecular basis for the role of nebulin as a thin filament "molecular ruler" and the impact of nemaline myopathies mutations that will aid future development of therapies.

biochemistry↗

bioRxiv2021

Rapid screening methods of potato cultivars for low glycemic traits

Potatoes are a dietary staple consumed by a significant portion of the world, providing valuable carbohydrates and vitamins with minimal fat. Most commercially produced potatoes have a high content of highly branched amylopectin starch, which generally results in a high glycemic index (GI). Consumption of foods with high levels of amylopectin elicit a rapid spike in blood glucose levels, which is undesirable for individuals who are pre-diabetic, diabetic, or obese. Some cultivars of potatoes with lower amylopectin levels have previously been identified and are commercially available in niche markets in some countries but are relatively unavailable in the United States and Latin America. Among Native communities in North America and in the high Andes countries of South America, some foods that include certain potato cultivars, may have been used to help people mitigate what is now defined as the effects of blood sugar and obesity. These cultivars are not widely available on a global market. This study utilizes three independent analyses of potato starch: microscopic examination of granule structure, water absorption, and spectrophotometry of iodine complexes to identify potato cultivars with low amylopectin that are thought to have low GI potential. Differences among cultivars tested were detected by all three types of analyses. Of the 60 potato cultivars evaluated the most promising are Huckleberry Gold, Muru, Multa, Green Mountain, and an October Blue x Colorado Rose cross. Further work is necessary to document the ability of these low amylopectin cultivars to reduce blood glucose spike levels in human subjects.

plant biology↗

bioRxiv2020

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

As the COVID-19 pandemic escalates, the need for effective vaccination programs, therapeutic intervention, and diagnostic tools increases. Here, we identified 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD) of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following a detailed epitope determination and characterization of the binding mode by structural analysis, we constructed a hetero-bivalent Nb targeting two different epitopes within the RBD:ACE2 interface. This resulted in a high-affinity binder with a viral neutralization efficacy in the picomolar range. Using the bivalent Nb as a surrogate, we established a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in the serum of convalescent or vaccinated patients. As demonstrated, the NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, helping to monitor immune status or guide vaccine design. This approach is easily transferrable to diagnostic laboratories worldwide.

immunology↗

bioRxiv2020

Molecular plasticity of the native mouse skeletal sarcomere revealed by cryo-ET

Sarcomeres are the force-generating and load-bearing devices of muscles. A precise molecular understanding of how the entire sarcomere is built is required to understand its role in health, disease and ageing. Here, we determine the in situ molecular architecture of vertebrate skeletal sarcomeres through electron cryo-tomography of cryo-focused ion beam-milled native myofibrils. The reconstructions reveal the three-dimensional organisation and interaction of actin and myosin filaments in the A-band, I-band and Z-disc and demonstrate how -actinin cross-links antiparallel actin filaments to form a mesh-like structure in the Z-disc at an unprecedented level of molecular detail. A prominent feature is a so-far undescribed doublet of -actinin cross-links with [~] 6 nm spacing. Sub-volume averaging shows the interaction between myosin, tropomyosin and actin in molecular detail at [~] 10 [A] resolution and reveals two coexisting conformations of actin-bound heads. The flexible orientation of the lever arm and the essential and regulatory light chains allow the two heads of the "double-headed" myosin not only to interact with the same actin filament but also to split between two actin filaments. Our results provide new insights into the conformational plasticity and fundamental organisation of vertebrate skeletal muscle and serve as a strong foundation for future in situ investigations of muscle diseases.

cell biology↗

bioRxiv2020

TranSPHIRE: Automated and feedback-optimized on-the-fly processing for cryo-EM

Single particle electron cryomicroscopy (cryo-EM) requires full automation to allow high-throughput structure determination which is especially important for drug discovery research. Although several software packages exist where parts of the cryo-EM pipeline are automated, a complete solution that offers reliable, quality-optimized on-the-fly processing, resulting in a high-resolution three-dimensional reconstruction does not exist. Here we present TranSPHIRE: A software package for fully automated processing of cryo-EM data sets during data acquisition. TranSPHIRE transfers data from the microscope, automatically applies the common pre-processing steps, picks particles, performs 2D clustering, and 3D refinement parallel to image recording. Importantly, TranSPHIRE introduces a machine learning-based feedback loop to re-train its internally used picking model to adapt to any given data set live during processing. This elegant approach enables TranSPHIRE to process data more effectively, producing high-quality particle stacks. TranSPHIRE collects, and displays all microscope settings and metrics generated by its individual tools, in order to allow users to quickly evaluate data during acquisition. TranSPHIRE can run on a single work station and also includes the automated processing of filaments.

biophysics↗

bioRxiv2020

Real-time biomedical knowledge synthesis of the exponentially growing world wide web using unsupervised neural networks

Decoding disease mechanisms for addressing unmet clinical need demands the rapid assimilation of the exponentially growing biomedical knowledge. These are either inherently unstructured and non-conducive to current computing paradigms or siloed into structured databases requiring specialized bioinformatics. Despite the recent renaissance in unsupervised neural networks for deciphering unstructured natural languages and the availability of numerous bioinformatics resources, a holistic platform for real-time synthesis of the scientific literature and seamless triangulation with deep omic insights and real-world evidence has not been advanced. Here, we introduce the nferX platform that makes the highly unstructured biomedical knowledge computable and supports the seamless visual triangulation with statistical inference from diverse structured databases. The nferX platform will accelerate and amplify the research potential of subject-matter experts as well as non-experts across the life science ecosystem (https://academia.nferx.com/).

bioinformatics↗

bioRxiv2020

Knowledge synthesis from 100 million biomedical documents augments the deep expression profiling of coronavirus receptors

The COVID-19 pandemic demands assimilation of all available biomedical knowledge to decode its mechanisms of pathogenicity and transmission. Despite the recent renaissance in unsupervised neural networks for decoding unstructured natural languages, a platform for the real-time synthesis of the exponentially growing biomedical literature and its comprehensive triangulation with deep omic insights is not available. Here, we present the nferX platform for dynamic inference from over 45 quadrillion possible conceptual associations extracted from unstructured biomedical text, and their triangulation with Single Cell RNA-sequencing based insights from over 25 tissues. Using this platform, we identify intersections between the pathologic manifestations of COVID-19 and the comprehensive expression profile of the SARS-CoV-2 receptor ACE2. We find that tongue keratinocytes, airway club cells, and ciliated cells are likely underappreciated targets of SARS-CoV-2 infection, in addition to type II pneumocytes and olfactory epithelial cells. We further identify mature small intestinal enterocytes as a possible hotspot of COVID-19 fecal-oral transmission, where an intriguing maturation-correlated transcriptional signature is shared between ACE2 and the other coronavirus receptors DPP4 (MERS-CoV) and ANPEP (-coronavirus). This study demonstrates how a holistic data science platform can leverage unprecedented quantities of structured and unstructured publicly available data to accelerate the generation of impactful biological insights and hypotheses. The nferX Platform Single-cell resource - https://academia.nferx.com/

genomics↗

bioRxiv2020

Two particle picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER

Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here we present two approaches for selecting filamentous complexes: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, the other one, called SPHIRE-STRIPER, is based on a classical line detection approach. The advantage of the crYOLO based procedure is that it accurately performs on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. We evaluate the performance of both procedures on tobacco mosaic virus and filamentous F-actin data sets to demonstrate the robustness of each method.

biophysics↗

bioRxiv2020

Chromatin modifiers and recombination factors promote a telomere fold-back structure, that is lost during replicative senescence

Telomeres adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomeric chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence. Author summaryTelomeres are the protective caps of chromosome ends and prevent the activation of a local DNA damage response. In many organisms, telomeres engage in a loop-like structure which may provide an additional layer of end protection. As we still lack insight into the regulation of the folded telomere structure, we used budding yeast to establish a method to measure telomere folding and then study the genetic requirements for its establishment. We found that cells require the homologous recombination machinery as well as components of the DNA damage checkpoint to successfully establish a folded telomere. Through the deletion of telomerase in budding yeast, we investigated how telomere folding was regulated during replicative senescence, a process that occurs in the majority of telomerase negative human cells. During senescence, telomeres gradually shorten and erode until cells stop dividing which is a potent tumor suppressor and prevents unscheduled growth of potential cancer cells. We found, that the folded telomere structure is compromised as part of the cellular senescence response, but not due to telomere shortening per se. We think, that an altered telomeric chromatin environment during senescence is important to maintain an open state - which may be important for signaling or for repair.

genetics↗