Publications by authors named "Michael Knop"

83 Publications

Up-regulation of ubiquitin-proteasome activity upon loss of NatA-dependent N-terminal acetylation.

Life Sci Alliance 2022 Feb 11;5(2). Epub 2021 Nov 11.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

N-terminal acetylation is a prominent protein modification, and inactivation of N-terminal acetyltransferases (NATs) cause protein homeostasis stress. Using multiplexed protein stability profiling with linear ubiquitin fusions as reporters for the activity of the ubiquitin proteasome system, we observed increased ubiquitin proteasome system activity in NatA, but not NatB or NatC mutants. We find several mechanisms contributing to this behavior. First, NatA-mediated acetylation of the N-terminal ubiquitin-independent degron regulates the abundance of Rpn4, the master regulator of the expression of proteasomal genes. Second, the abundance of several E3 ligases involved in degradation of UFD substrates is increased in cells lacking NatA. Finally, we identify the E3 ligase Tom1 as a novel chain-elongating enzyme (E4) involved in the degradation of linear ubiquitin fusions via the formation of branched K11, K29, and K48 ubiquitin chains, independently of the known E4 ligases involved in UFD, leading to enhanced ubiquitination of the UFD substrates.
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http://dx.doi.org/10.26508/lsa.202000730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605321PMC
February 2022

Sex without crossing over in the yeast Saccharomycodes ludwigii.

Genome Biol 2021 11 3;22(1):303. Epub 2021 Nov 3.

Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

Background: Intermixing of genomes through meiotic reassortment and recombination of homologous chromosomes is a unifying theme of sexual reproduction in eukaryotic organisms and is considered crucial for their adaptive evolution. Previous studies of the budding yeast species Saccharomycodes ludwigii suggested that meiotic crossing over might be absent from its sexual life cycle, which is predominated by fertilization within the meiotic tetrad.

Results: We demonstrate that recombination is extremely suppressed during meiosis in Sd. ludwigii. DNA double-strand break formation by the conserved transesterase Spo11, processing and repair involving interhomolog interactions are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers in its genome. A strong AT bias of spontaneous mutations and the absence of recombination are likely responsible for its unusually low genomic GC level.

Conclusions: Sd. ludwigii has followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent mating between products of the same meiotic event with the extreme suppression of meiotic recombination. This life style ensures preservation of heterozygosity throughout its genome and may enable the species to adapt to its environment and survive with only minimal levels of rare meiotic recombination. We propose Sd. ludwigii as an excellent natural forum for the study of genome evolution and recombination rates.
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http://dx.doi.org/10.1186/s13059-021-02521-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8567612PMC
November 2021

The NADPH Oxidase A of Is Essential for Pathogenicity, Normal Development, and Stress Tolerance, and It Interacts with Yap1 to Regulate Redox Homeostasis.

J Fungi (Basel) 2021 Sep 9;7(9). Epub 2021 Sep 9.

Department of Genetics & Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, 15784 Athens, Greece.

Maintenance of redox homeostasis is vital for aerobic organisms and particularly relevant to plant pathogens. A balance is required between their endogenous ROS production, which is important for their development and pathogenicity, and host-derived oxidative stress. Endogenous ROS in fungi are generated by membrane-bound NADPH oxidase (NOX) complexes and the mitochondrial respiratory chain, while transcription factor Yap1 is a major regulator of the antioxidant response. Here, we investigated the roles of NoxA and Yap1 in fundamental biological processes of the important plant pathogen . Deletion of impaired growth and morphogenesis, compromised formation of hyphopodia, diminished penetration ability and pathogenicity, increased sensitivity against antifungal agents, and dysregulated expression of antioxidant genes. On the other hand, deletion of resulted in defects in conidial and microsclerotia formation, increased sensitivity against oxidative stress, and down-regulated antioxidant genes. Localized accumulation of ROS was observed before conidial fusion and during the heterokaryon incompatibility reaction upon nonself fusion. The frequency of inviable fusions was not affected by the deletion of Yap1. Analysis of a double knockout mutant revealed an epistatic relationship between and . Our results collectively reveal instrumental roles of NoxA and ROS homeostasis in the biology of .
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http://dx.doi.org/10.3390/jof7090740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8468606PMC
September 2021

α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.

Cells 2021 08 28;10(9). Epub 2021 Aug 28.

Department of Molecular Microbiology and Genetics, Institute for Microbiology and Genetics, University of Göttingen, 37077 Göttingen, Germany.

Parkinson's disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.
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http://dx.doi.org/10.3390/cells10092229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8468666PMC
August 2021

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): study protocol for a two-factorial randomized controlled multi-arm trial with cluster sampling.

Trials 2021 Sep 26;22(1):656. Epub 2021 Sep 26.

Division of Clinical Tropical Medicine, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

Background: To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic.

Methods: This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals' self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups (individuals vs. all household members) and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate.

Discussion: Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures.

Trial Registration: ClinicalTrials.gov DRKS00023271 . Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien).
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http://dx.doi.org/10.1186/s13063-021-05619-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8474710PMC
September 2021

Investigating the Use of Telemedicine for Digitally Mediated Delegation in Team-Based Primary Care: Mixed Methods Study.

J Med Internet Res 2021 08 26;23(8):e28151. Epub 2021 Aug 26.

Chair of Information Systems, University of Siegen, Siegen, Germany.

Background: Owing to the shortage of medical professionals, as well as demographic and structural challenges, new care models have emerged to find innovative solutions to counter medical undersupply. Team-based primary care using medical delegation appears to be a promising approach to address these challenges; however, it demands efficient communication structures and mechanisms to reinsure patients and caregivers receive a delegated, treatment-related task. Digital health care technologies hold the potential to render these novel processes effective and demand driven.

Objective: The goal of this study is to recreate the daily work routines of general practitioners (GPs) and medical assistants (MAs) to explore promising approaches for the digital moderation of delegation processes and to deepen the understanding of subjective and perceptual factors that influence their technology assessment and use.

Methods: We conducted a combination of 19 individual and group interviews with 12 GPs and 14 MAs, seeking to identify relevant technologies for delegation purposes as well as stakeholders' perceptions of their effectiveness. Furthermore, a web-based survey was conducted asking the interviewees to order identified technologies based on their assessed applicability in multi-actor patient care. Interview data were analyzed using a three-fold inductive coding procedure. Multidimensional scaling was applied to analyze and visualize the survey data, leading to a triangulation of the results.

Results: Our results suggest that digital mediation of delegation underlies complex, reciprocal processes and biases that need to be identified and analyzed to improve the development and distribution of innovative technologies and to improve our understanding of technology use in team-based primary care. Nevertheless, medical delegation enhanced by digital technologies, such as video consultations, portable electrocardiograms, or telemedical stethoscopes, can counteract current challenges in primary care because of its unique ability to ensure both personal, patient-centered care for patients and create efficient and needs-based treatment processes.

Conclusions: Technology-mediated delegation appears to be a promising approach to implement innovative, case-sensitive, and cost-effective ways to treat patients within the paradigm of primary care. The relevance of such innovative approaches increases with the tremendous need for differentiated and effective care, such as during the ongoing COVID-19 pandemic. For the successful and sustainable adoption of innovative technologies, MAs represent essential team members. In their role as mediators between GPs and patients, MAs are potentially able to counteract patients' resistance toward using innovative technology and compensate for patients' limited access to technology and care facilities.
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http://dx.doi.org/10.2196/28151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8430853PMC
August 2021

Starvation-induced cell fusion and heterokaryosis frequently escape imperfect allorecognition systems in an asexual fungal pathogen.

BMC Biol 2021 08 24;19(1):169. Epub 2021 Aug 24.

Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

Background: Asexual fungi include important pathogens of plants and other organisms, and their effective management requires understanding of their evolutionary dynamics. Genetic recombination is critical for adaptability and could be achieved via heterokaryosis - the co-existence of genetically different nuclei in a cell resulting from fusion of non-self spores or hyphae - and the parasexual cycle in the absence of sexual reproduction. Fusion between different strains and establishment of viable heterokaryons are believed to be rare due to non-self recognition systems. Here, we investigate the extent and mechanisms of cell fusion and heterokaryosis in the important asexual plant pathogen Verticillium dahliae.

Results: We used live-cell imaging and genetic complementation assays of tagged V. dahliae strains to analyze the extent of non-self vegetative fusion, heterokaryotic cell fate, and nuclear behavior. An efficient CRISPR/Cas9-mediated system was developed to investigate the involvement of autophagy in heterokaryosis. Under starvation, non-self fusion of germinating spores occurs frequently regardless of the previously assessed vegetative compatibility of the partners. Supposedly "incompatible" fusions often establish viable heterokaryotic cells and mosaic mycelia, where nuclei can engage in fusion or transfer of genetic material. The molecular machinery of autophagy has a protective function against the destruction of "incompatible" heterokaryons.

Conclusions: We demonstrate an imperfect function of somatic incompatibility systems in V. dahliae. These systems frequently tolerate the establishment of heterokaryons and potentially the initiation of the parasexual cycle even between strains that were previously regarded as "incompatible."
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http://dx.doi.org/10.1186/s12915-021-01101-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385987PMC
August 2021

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Mol Cell 2021 06 10;81(11):2460-2476.e11. Epub 2021 May 10.

Institute of Molecular Biology (IMB), Mainz, Germany. Electronic address:

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
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http://dx.doi.org/10.1016/j.molcel.2021.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189435PMC
June 2021

Colorimetric RT-LAMP and LAMP-sequencing forDetecting SARS-CoV-2 RNA in Clinical Samples.

Bio Protoc 2021 Mar 20;11(6):e3964. Epub 2021 Mar 20.

Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi , 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.
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http://dx.doi.org/10.21769/BioProtoc.3964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032487PMC
March 2021

Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions.

Curr Genet 2021 Jun 13;67(3):471-485. Epub 2021 Feb 13.

Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.
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http://dx.doi.org/10.1007/s00294-021-01157-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139932PMC
June 2021

N-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles in Saccharomyces cerevisiae.

Cell Rep 2021 02;34(5):108711

Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg 69120, Germany. Electronic address:

N-terminal (Nt) acetylation is a highly prevalent co-translational protein modification in eukaryotes, catalyzed by at least five Nt acetyltransferases (Nats) with differing specificities. Nt acetylation has been implicated in protein quality control, but its broad biological significance remains elusive. We investigate the roles of the two major Nats of S. cerevisiae, NatA and NatB, by performing transcriptome, translatome, and proteome profiling of natAΔ and natBΔ mutants. Our results reveal a range of NatA- and NatB-specific phenotypes. NatA is implicated in systemic adaptation control, because natAΔ mutants display altered expression of transposons, sub-telomeric genes, pheromone response genes, and nuclear genes encoding mitochondrial ribosomal proteins. NatB predominantly affects protein folding, because natBΔ mutants, to a greater extent than natA mutants, accumulate protein aggregates, induce stress responses, and display reduced fitness in the absence of the ribosome-associated chaperone Ssb. These phenotypic differences indicate that controlling Nat activities may serve to elicit distinct cellular responses.
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http://dx.doi.org/10.1016/j.celrep.2021.108711DOI Listing
February 2021

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): a structured summary of a study protocol for a cluster-randomised, two-factorial controlled trial.

Trials 2021 Jan 8;22(1):39. Epub 2021 Jan 8.

Section Clinical Tropical Medicine, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

Objectives: In this cluster-randomised controlled study (CoV-Surv Study), four different "active" SARS-CoV-2 testing strategies for general population surveillance are evaluated for their effectiveness in determining and predicting the prevalence of SARS-CoV-2 infections in a given population. In addition, the costs and cost-effectiveness of the four surveillance strategies will be assessed. Further, this trial is supplemented by a qualitative component to determine the acceptability of each strategy. Findings will inform the choice of the most effective, acceptable and affordable strategy for SARS-CoV-2 surveillance, with the most effective and cost-effective strategy becoming part of the local public health department's current routine health surveillance activities. Investigating its everyday performance will allow us to examine the strategy's applicability to real time prevalence prediction and the usefulness of the resulting information for local policy makers to implement countermeasures that effectively prevent future nationwide lockdowns. The authors would like to emphasize the importance and relevance of this study and its expected findings in the context of population-based disease surveillance, especially in respect to the current SARS-CoV-2 pandemic. In Germany, but also in many other countries, COVID-19 surveillance has so far largely relied on passive surveillance strategies that identify individuals with clinical symptoms, monitor those cases who then tested positive for the virus, followed by tracing of individuals in close contact to those positive cases. To achieve higher effectiveness in population surveillance and to reliably predict the course of an outbreak, screening and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. To better control and manage the SARS-CoV-2 pandemic, current strategies therefore need to be complemented by an active surveillance of the wider population, i.e. routinely conducted testing and monitoring activities to identify and isolate infected individuals regardless of their clinical symptoms. Such active surveillance strategies will enable more effective prevention of the spread of the virus as they can generate more precise population-based parameters during a pandemic. This essential information will be required in order to determine the best strategic and targeted short-term countermeasures to limit infection spread locally.

Trial Design: This trial implements a cluster-randomised, two-factorial controlled, prospective, interventional, single-blinded design with four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy.

Participants: Eligible are individuals age 7 years or older living in Germany's Rhein-Neckar Region who consent to provide a saliva sample (all four arms) after completion of a brief questionnaire (two arms only). For the qualitative component, different samples of study participants and non-participants (i.e. eligible for study, but refuse to participate) will be identified for additional interviews. For these interviews, only individuals age 18 years or older are eligible.

Intervention And Comparator: Of the four surveillance strategies to be assessed and compared, Strategy A1 is considered the gold standard for prevalence estimation and used to determine bias in other arms. To determine the cost-effectiveness, each strategy is compared to status quo, defined as the currently practiced passive surveillance approach. Strategy A1: Individuals (one per household) receive information and study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the sample is tested for SARS-CoV-2 using Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP). Strategy A2: Individuals (one per household) receive information and study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. Strategy B1: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms and risk exposures. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the saliva sample is tested for SARS-CoV-2 using RT-LAMP. Strategy B2: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. In each strategy, RT-LAMP positive samples are additionally analyzed with qPCR in order to minimize the number of false positives.

Main Outcomes: The identification of the one best strategy will be determined by a set of parameters. Primary outcomes include costs per correctly screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Secondary outcomes include participation rate, costs per asymptomatic case, prevalence estimates, number of asymptomatic cases per study arm, ratio of symptomatic to asymptomatic cases per study arm, participant satisfaction. Additional study components (not part of the trial) include cost effectiveness of each of the four surveillance strategies compared to passive monitoring (i.e. status quo), development of a prognostic model to predict hospital utilization caused by SARS-CoV-2, time from test shipment to test application and time from test shipment to test result, and perception and preferences of the persons to be tested with regard to test strategies.

Randomisation: Samples are drawn in three batches of three continuous weeks. Randomisation follows a two-stage process. First, a total of 220 sampling points have been allocated to the three different batches. To obtain an integer solution, the Cox-algorithm for controlled rounding has been used. Afterwards, sample points have been drawn separately per batch, following a probability proportional to size (PPS) random sample. Second, for each cluster the same number of residential addresses is randomly sampled from the municipal registries (self-weighted sample of individuals). The 28,125 addresses drawn per municipality are then randomly allocated to the four study arms A1, A2, B1, and B2 in the ratio 5 to 2.5 to 14 to 7 based on the expected response rates in each arm and the sensitivity and specificity of the pre-screening tool as applied in strategy B1 and B2. Based on the assumptions, this allocation should yield 2500 saliva samples in each strategy. Although a municipality can be sampled by multiple batches and the overall number of addresses per municipality might vary, the number of addresses contacted in each arm is kept constant.

Blinding (masking): The design is single-blinded, meaning the staff conducting the SARS-CoV-2 tests are unaware of the study arm assignment of each single participant and test sample.

Sample Sizes: Total sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (i.e. 2,500 participants per arm). For the qualitative component, up to 60 in-depth interviews will be conducted with about 30 study participants (up to 15 in each arm A and B) and 30 participation refusers (up to 15 in each arm A and B) purposefully selected from the quantitative study sample to represent a variety of gender and ages to explore experiences with admission or rejection of study participation. Up to 25 asymptomatic SARS-CoV-2 positive study participants will be purposefully selected to explore the way in which asymptomatic men and women diagnosed with SARS-CoV-2 give meaning to their diagnosis and to the dialectic between feeling concurrently healthy and yet also being at risk for transmitting COVID-19. In addition, 100 randomly selected study participants will be included to explore participants' perspective on testing processes and implementation.

Trial Status: Final protocol version is "Surveillance_Studienprotokoll_03Nov2020_v1_2" from November 3, 2020. Recruitment started November 18, 2020 and is expected to end by or before December 31, 2020.

Trial Registration: The trial is currently being registered with the German Clinical Trials Register (Deutsches Register Klinischer Studien), DRKS00023271 ( https://www.drks.de/drks_web/navigate.do?navigationId=trial . HTML&TRIAL_ID=DRKS00023271). Retrospectively registered 30 November 2020.

Full Protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
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http://dx.doi.org/10.1186/s13063-020-04982-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791150PMC
January 2021

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

iScience 2021 Jan 7;24(1):101895. Epub 2020 Dec 7.

Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany.

Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.
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http://dx.doi.org/10.1016/j.isci.2020.101895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753144PMC
January 2021

Hex1, the Major Component of Woronin Bodies, Is Required for Normal Development, Pathogenicity, and Stress Response in the Plant Pathogenic Fungus .

J Fungi (Basel) 2020 Dec 7;6(4). Epub 2020 Dec 7.

Department of Genetics & Biotechnology, Faculty of Biology, National and Kapodistrian University of Athens, 15784 Athens, Greece.

Woronin bodies are membrane-bound organelles of filamentous ascomycetes that mediate hyphal compartmentalization by plugging septal pores upon hyphal damage. Their major component is the peroxisomal protein Hex1, which has also been implicated in additional cellular processes in fungi. Here, we analyzed the Hex1 homolog of , an important asexual plant pathogen, and we report its pleiotropic involvement in fungal growth, physiology, stress response, and pathogenicity. Alternative splicing of the gene can lead to the production of two Hex1 isoforms, which are structurally similar to their homolog. We show that Hex1 is targeted to the septum, consistently with its demonstrated function in sealing hyphal compartments to prevent excessive cytoplasmic bleeding upon injury. Furthermore, our investigation provides direct evidence for significant contributions of Hex1 in growth and morphogenesis, as well as in asexual reproduction capacity. We discovered that Hex1 is required both for normal responses to osmotic stress and factors that affect the cell wall and plasma-membrane integrity, and for normal resistance to oxidative stress and reactive oxygen species (ROS) homeostasis. The mutant exhibited diminished ability to colonize and cause disease on eggplant. Overall, we show that Hex1 has fundamentally important multifaceted roles in the biology of .
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http://dx.doi.org/10.3390/jof6040344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762394PMC
December 2020

Extensive 5'-surveillance guards against non-canonical NAD-caps of nuclear mRNAs in yeast.

Nat Commun 2020 11 2;11(1):5508. Epub 2020 Nov 2.

Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, 69120, Heidelberg, Germany.

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%). NAD incorporation occurs mainly during transcription initiation by RNA polymerase II, which uses distinct promoters with a YAAG core motif for this purpose. Most NAD-RNAs are 3'-truncated. At least three decapping enzymes, Rai1, Dxo1, and Npy1, guard against NAD-RNA at different cellular locations, targeting overlapping transcript populations. NAD-mRNAs are not translatable in vitro. Our work indicates that in budding yeast, most of the NAD incorporation into RNA seems to be disadvantageous to the cell, which has evolved a diverse surveillance machinery to prematurely terminate, decap and reject NAD-RNAs.
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http://dx.doi.org/10.1038/s41467-020-19326-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606564PMC
November 2020

Investigating the Acceptance of Video Consultation by Patients in Rural Primary Care: Empirical Comparison of Preusers and Actual Users.

JMIR Med Inform 2020 Oct 22;8(10):e20813. Epub 2020 Oct 22.

Department of General Practice and Family Medicine, Philipps-University, Marburg, Germany.

Background: The ongoing digitalization in health care is enabling patients to receive treatment via telemedical technologies, such as video consultation (VC), which are increasingly being used by general practitioners. Rural areas in particular exhibit a rapidly aging population, with an increase in associated health issues, whereas the level of attraction for working in those regions is decreasing for young physicians. Integrating telemedical approaches in treating patients can help lessen the professional workload and counteract the trend toward the spatial undersupply in many countries. As a result, an increasing number of patients are being confronted with digital treatment and new forms of care delivery. These novel ways of care engender interactions with patients and their private lives in unprecedented ways, calling for studies that incorporate patient needs, expectations, and behavior into the design and application of telemedical technology within the field of primary care.

Objective: This study aims to unveil and compare the acceptance-promoting factors of patients without (preusers) and with experiences (actual users) in using VC in a primary care setting and to provide implications for the design, theory, and use of VC.

Methods: In total, 20 semistructured interviews were conducted with patients in 2 rural primary care practices to identify and analyze patient needs, perceptions, and experiences that facilitate the acceptance of VC technology and adoption behavior. Both preusers and actual users of VC were engaged, allowing for an empirical comparison. For data analysis, a procedure was followed based on open, axial, and selective coding.

Results: The study delivers factors and respective subdimensions that foster the perceptions of patients toward VC in rural primary care. Factors cover attitudes and expectations toward the use of VC, the patient-physician relationship and its impact on technology assessment and use, patients' rights and obligations that emerge with the introduction of VC in primary care, and the influence of social norms on the use of VC and vice versa. With regard to these factors, the results indicate differences between preusers and actual users of VC, which imply ways of designing and implementing VC concerning the respective user group. Actual users attach higher importance to the perceived benefits of VC and their responsibility to use it appropriately, which might be rooted in the technological intervention they experienced. On the contrary, preusers valued the opinions and expectations of their peers.

Conclusions: The way the limitations and potential of VC are perceived varies across patients. When practicing VC in primary care, different aspects should be considered when dealing with preusers, such as maintaining a physical interaction with the physician or incorporating social cues. Once the digital intervention takes place, patients tend to value benefits such as flexibility and effectiveness over potential concerns.
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http://dx.doi.org/10.2196/20813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644376PMC
October 2020

Single-Color Fluorescence Lifetime Cross-Correlation Spectroscopy In Vivo.

Biophys J 2020 10 20;119(7):1359-1370. Epub 2020 Aug 20.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Heidelberg, Germany; Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany. Electronic address:

The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.
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http://dx.doi.org/10.1016/j.bpj.2020.06.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568003PMC
October 2020

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Viruses 2020 08 7;12(8). Epub 2020 Aug 7.

Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.
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http://dx.doi.org/10.3390/v12080863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472728PMC
August 2020

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

Sci Transl Med 2020 08 27;12(556). Epub 2020 Jul 27.

Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.
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http://dx.doi.org/10.1126/scitranslmed.abc7075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574920PMC
August 2020

Exploring whole-genome duplicate gene retention with complex genetic interaction analysis.

Science 2020 06;368(6498)

Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors.
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http://dx.doi.org/10.1126/science.aaz5667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539174PMC
June 2020

CRISPR-Cas12a-assisted PCR tagging of mammalian genes.

J Cell Biol 2020 06;219(6)

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), German Cancer Research Center (DKFZ)-ZMBH Alliance, Heidelberg, Germany.

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
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http://dx.doi.org/10.1083/jcb.201910210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265327PMC
June 2020

CDK1 couples proliferation with protein synthesis.

J Cell Biol 2020 03;219(3)

Division of Biochemistry, Mannheim Institute for Innate Immunoscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.
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http://dx.doi.org/10.1083/jcb.201906147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054999PMC
March 2020

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast.

EMBO J 2020 01 5;39(2):e102586. Epub 2019 Dec 5.

DKFZ-ZMBH Alliance and CellNetworks Cluster of Excellence, Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.
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http://dx.doi.org/10.15252/embj.2019102586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960443PMC
January 2020

Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast.

Nat Commun 2019 07 4;10(1):2960. Epub 2019 Jul 4.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120, Heidelberg, Germany.

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.
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http://dx.doi.org/10.1038/s41467-019-10816-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609715PMC
July 2019

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Elife 2019 06 7;8. Epub 2019 Jun 7.

Centre for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.
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http://dx.doi.org/10.7554/eLife.45506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586462PMC
June 2019

Self-Learning Microfluidic Platform for Single-Cell Imaging and Classification in Flow.

Micromachines (Basel) 2019 May 9;10(5). Epub 2019 May 9.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Universität Heidelberg, 69120 Heidelberg, Germany.

Single-cell analysis commonly requires the confinement of cell suspensions in an analysis chamber or the precise positioning of single cells in small channels. Hydrodynamic flow focusing has been broadly utilized to achieve stream confinement in microchannels for such applications. As imaging flow cytometry gains popularity, the need for imaging-compatible microfluidic devices that allow for precise confinement of single cells in small volumes becomes increasingly important. At the same time, high-throughput single-cell imaging of cell populations produces vast amounts of complex data, which gives rise to the need for versatile algorithms for image analysis. In this work, we present a microfluidics-based platform for single-cell imaging in-flow and subsequent image analysis using variational autoencoders for unsupervised characterization of cellular mixtures. We use simple and robust Y-shaped microfluidic devices and demonstrate precise 3D particle confinement towards the microscope slide for high-resolution imaging. To demonstrate applicability, we use these devices to confine heterogeneous mixtures of yeast species, brightfield-image them in-flow and demonstrate fully unsupervised, as well as few-shot classification of single-cell images with 88% accuracy.
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http://dx.doi.org/10.3390/mi10050311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563144PMC
May 2019

YeastRGB: comparing the abundance and localization of yeast proteins across cells and libraries.

Nucleic Acids Res 2019 01;47(D1):D1245-D1249

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.

The ability to measure the abundance and visualize the localization of proteins across the yeast proteome has stimulated hypotheses on gene function and fueled discoveries. While the classic C' tagged GFP yeast library has been the only resource for over a decade, the recent development of the SWAT technology has led to the creation of multiple novel yeast libraries where new-generation fluorescent reporters are fused at the N' and C' of open reading frames. Efficient access to these data requires a user interface to visualize and compare protein abundance, localization and co-localization across cells, strains, and libraries. YeastRGB (www.yeastRGB.org) was designed to address such a need, through a user-friendly interface that maximizes informative content. It employs a compact display where cells are cropped and tiled together into a 'cell-grid.' This representation enables viewing dozens of cells for a particular strain within a display unit, and up to 30 display units can be arrayed on a standard high-definition screen. Additionally, the display unit allows users to control zoom-level and overlay of images acquired using different color channels. Thus, YeastRGB makes comparing abundance and localization efficient, across thousands of cells from different strains and libraries.
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http://dx.doi.org/10.1093/nar/gky941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324022PMC
January 2019

Monitoring Protein Dynamics in Protein -Mannosyltransferase Mutants In Vivo by Tandem Fluorescent Protein Timers.

Molecules 2018 Oct 12;23(10). Epub 2018 Oct 12.

Centre for Organismal Studies (COS), Heidelberg University, 69120 Heidelberg, Germany.

For proteins entering the secretory pathway, a major factor contributing to maturation and homeostasis is glycosylation. One relevant type of protein glycosylation is -mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism revealed that more than 26% of all proteins entering the secretory pathway receive -mannosyl glycans. In a first attempt to understand the impact of -mannosylation on these proteins, we took advantage of a tandem fluorescent timer (tFT) reporter to monitor different aspects of protein dynamics. We analyzed tFT-reporter fusions of 137 unique -mannosylated proteins, mainly of the secretory pathway and the plasma membrane, in mutants lacking the major protein -mannosyltransferases Pmt1, Pmt2, or Pmt4. In these three Δ mutants, a total of 39 individual proteins were clearly affected, and Pmt-specific substrate proteins could be identified. We observed that -mannosylation may cause both enhanced and diminished protein abundance and/or stability when compromised, and verified our findings on the examples of Axl2-tFT and Kre6-tFT fusion proteins. The identified target proteins are a valuable resource towards unraveling the multiple functions of -mannosylation at the molecular level.
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http://dx.doi.org/10.3390/molecules23102622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222916PMC
October 2018

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis.

Mol Syst Biol 2018 09 4;14(9):e8355. Epub 2018 Sep 4.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, University of Heidelberg, Heidelberg, Germany

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior-posterior axis in the early embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121778PMC
http://dx.doi.org/10.15252/msb.20188355DOI Listing
September 2018

Genome-wide C-SWAT library for high-throughput yeast genome tagging.

Nat Methods 2018 08 9;15(8):598-600. Epub 2018 Jul 9.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
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http://dx.doi.org/10.1038/s41592-018-0045-8DOI Listing
August 2018
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