Biology subjects

Müller, J.

Publications and source records attributed to Müller, J..

26 records · Page 2Linked to original sources

bioRxiv2023

Complex genomic landscape of inversion polymorphism in Europe's most destructive forest pest

In many species, polymorphic inversions underlie complex phenotypic polymorphisms and facilitate local adaptation in the face of gene flow. Multiple polymorphic inversions can co-occur in a genome, but the prevalence, evolutionary significance, and limits to complexity of genomic inversion landscapes remain poorly understood. Here, we examine genome-wide variation in one of Europes most destructive forest pests, the spruce bark beetle Ips typographus, scan for polymorphic inversions, and test whether inversions are involved in key adaptations in this species. We analyzed 240 individuals from 18 populations across the species European range and, using a whole-genome resequencing approach, identified 27 polymorphic inversions covering approximately 28% of the genome. The inversions vary in size and in levels of intra-inversion recombination, are highly polymorphic across the species range, and often overlap, forming a complex genomic architecture. We test several mechanisms, including directional selection, overdominance and associative overdominance that can contribute to the maintenance of inversion polymorphisms in the genome. We show that the heterogeneous inversion landscape is likely maintained by the combined action of several evolutionary forces and that inversions are enriched in odorant receptor genes encoding key elements of recognition pathways for host plants, mates, and symbiotic fungi. Our results indicate that the genome of this major forest pest of growing social, political, and economic importance harbors one of the most complex inversion landscapes described to date posing a question about limits of genomic architecture complexity.

evolutionary biology↗

bioRxiv2022

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9

Herein, we developed a single and a duplex TaqMan quantitative qPCR for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wt-dhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

molecular biology↗

bioRxiv2021

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and channel substrates to the proteasome. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase resulting in co-degradation of separase along with securin. If true, this establishes Cdc48 as a key regulator of mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

cell biology↗

bioRxiv2021

Climate-induced forest dieback drives compositional change in insect communities that is concentrated amongst rare species

Marked decline in insect species richness, abundance and biomass have recently been quantified in Europe. We metabarcoded 224 Malaise-trap samples to investigate whether drought-induced forest dieback and subsequent salvage logging have an impact on flying insects (ca. 3000 insect species) in silver fir Pyrenean forests. We found no evidence that climate-induced forest dieback impacted species richness of flying insects but revealed compositional turnover patterns consistent with those seen during natural forest succession, given that the key covariates explaining compositional variation were canopy openness versus microhabitat diversity and deadwood amount at local and landscape scales, respectively. Importantly, most change was driven by rare species. In contrast, observed levels of salvage logging did not explain change in species richness or composition. Hence, although forest dieback appears to cause changes in species assemblages mimicking natural forest succession, it also increases the risk of catastrophic loss of rare species through homogenization of environmental conditions.

ecology↗

bioRxiv2020

Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1

The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated degradation (ERAD) are two essential components of the quality control system for proteins in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an ERAD substrate, indicating that the UPR and ERAD regulate each other. Viruses are intracellular parasites that exploit the host cell for their own benefit. Cytomegaloviruses selectively modulate the UPR to take advantage of beneficial and inhibit detrimental effects on viral replication. We have previously shown that murine and human cytomegaloviruses express homologous proteins (M50 and UL50, respectively) that dampen the UPR at late times post infection by inducing IRE1 degradation. However, the degradation mechanism has remained uncertain. Here we show that the cytomegalovirus M50 protein mediates IRE1 degradation by the proteasome. M50-dependent IRE1 degradation can be blocked by pharmacological inhibition of p97/VCP or by genetic ablation of SEL1L, both of which are component of the ERAD machinery. SEL1L acts as a cofactor of the E3 ubiquitin ligase HRD1, while p97/VCP is responsible for the extraction of ubiquitylated proteins from the ER to the cytosol. We further show that M50 facilitates the IRE1-SEL1L interaction by binding to both, IRE1 and SEL1L. These results indicate that the viral M50 protein dampens the UPR by tethering IRE1 to SEL1L, thereby promoting its degradation by the ERAD machinery. ImportanceViruses infect cells of their host and force them to produce virus progeny. This can impose stress on the host cell and activate counter-regulatory mechanisms. Protein overload in the endoplasmic reticulum (ER) leads to ER stress and triggers the unfolded protein response, which in turn upregulates protein folding and increases the degradation of proteins in the ER. Previous work has shown that cytomegaloviruses interfere with the unfolded protein response by degrading the sensor molecule IRE1. Herein we demonstrate how the viral M50 protein exploits the ER-associated degradation machinery to dispose of IRE1. Degradation of IRE1 curbs the unfolded protein response and helps the virus to increase the synthesis of its own proteins.

microbiology↗

bioRxiv2020

Structural basis for decoding active histone methylation marks by Polycomb Repressive Complex 2

Repression of genes by Polycomb requires that PRC2 modifies their chromatin by trimethylating lysine 27 on histone H3 (H3K27me3). At transcriptionally active genes, di- and trimethylated H3K36 inhibit PRC2. Here, the cryo-EM structure of PRC2 on dinucleosomes reveals how binding of its catalytic subunit EZH2 to nucleosomal DNA orients the H3 N-terminus via an extended network of interactions to place H3K27 into the active site. Unmodified H3K36 occupies a critical position in the EZH2-DNA interface. Mutation of H3K36 to arginine or alanine inhibits H3K27 methylation by PRC2 on nucleosomes in vitro. Accordingly, Drosophila H3K36A and H3K36R mutants show reduced levels of H3K27me3 and defective Polycomb repression of HOX genes. The relay of interactions between EZH2, the nucleosomal DNA and the H3 N-terminus therefore creates the geometry that permits allosteric inhibition of PRC2 by methylated H3K36 in transcriptionally active chromatin.

biochemistry↗

bioRxiv2019

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions including cell motility, signal transduction, and virus replication. Here we describe a dual color (DC) extension of the fluorescence z-scan technique which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the co-expression of a second distinctly colored fluorescent protein provides a soluble reference species, which delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2 but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ~16 M and Hill coefficient of n ~2.\n\nSIGNIFICANCEProtein binding to the plasma membrane of cells plays an important role in a multitude of cell functions and disease processes. Quantitative binding studies of protein/membrane interactions are almost exclusively limited to in vitro systems and may produce results that poorly mimic the authentic interactions in living cells. We report quantitative measurements of plasma membrane binding directly in living cells by using dual color z-scan fluorescence, which improves the detection threshold by an order of magnitude compared to our previous single color technique. This advance allowed us to examine the role of mutations on binding affinity and identify the presence of cooperative binding in protein systems with relevance to HIV/AIDS and cancer biology.

biophysics↗

bioRxiv2019

Doberman pinschers present autoimmunity associated with functional autoantibodies: a model to study the autoimmune background of human dilated cardiomyopathy

BackgroundAutoimmunity associated with autoantibodies directed against the {beta}1-adrenergic receptor ({beta}1-AAB) is increasingly accepted as driving human dilated cardiomyopathy (DCM). Unfortunately, animal models of DCM are lacking, preventing our knowledge about {beta}1-AAB autoimmunity in DCM from being extended and hindering the development of related treatment strategies.\n\nObjectivesTo introduce an animal model, we studied Doberman pinschers, which develop cardiomyopathy (DoCM), with similarities to human DCM, with regard to their {beta}1-AAB autoimmunity.\n\nMethodsEighty-seven DP with DoCM and 31 (at enrolment) healthy controls were analyzed for {beta}1-AAB; the receptor binding site and sensitivity to inhibition were determined. In controls who developed cardiomyopathy during the follow-up, {beta}1-AAB were analyzed during the DoCM progress.\n\nResultsFifty-nine (67.8%) DoCM dogs and 19 (61.3%) controls were {beta}1-AAB positive. Excluding the 9 controls who developed DoCM in the follow-up, {beta}1-AAB positivity tended to be more pronounced in DoCM.\n\nFrom the controls who developed DoCM, 8 were {beta}1-AAB positive (p=0.044 vs. dogs remaining healthy); their {beta}1-AAB level increased with the cardiomyopathy progress. Overall mortality and mortality exclusively due to cardiac reasons during the study period, were higher (p=0.002; p=0037) in {beta}1-AAB positive dogs. The dogs {beta}1-AAB targeted a specific epitope centralized on the second extracellular receptor and were sensitive to inhibition by drugs already successful tested for the corresponding human autoantibody.\n\nConclusionsDoberman pinschers presented {beta}1-AAB associated autoimmunity similar to that driving the pathogenesis of human DCM. Consequently, DP could remove the lack of animal models available for studying {beta}1-AAB autoimmunity in DCM.

pathology↗