Biology subjects

Mueller, F.

Publications and source records attributed to Mueller, F..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

ReSurveyGermany: Vegetation-plot time-series over the past hundred years in Germany

Vegetation-plot resurvey data are a main source of information on terrestrial biodiversity change, with records reaching back more than one century. Although more and more data from re-sampled plots have been published, there is not yet a comprehensive open-access dataset available for analysis. Here, we compiled and harmonised vegetation-plot resurvey data from Germany covering almost 100 years. We show the distribution of the plot data in space, time and across habitat types of the European Nature Information System (EUNIS). In addition, we include metadata on geographic location, plot size and vegetation structure. The data allow calculating temporal biodiversity change at the community scale and reach back further into the past than most comparable data yet available. They also enable tracking changes in the incidence and distribution of individual species across Germany. In summary, the data come at a level of detail that holds promise for broadening our understanding of the mechanisms and drivers behind plant diversity change over the last century.

ecology↗

bioRxiv2022

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus-end directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. This mechanism may be paradigmatic for Dynein activation by other adaptors at various cellular locales.

biochemistry↗

bioRxiv2022

Excessive self-grooming of Shank3 mutant mice is associated with gene dysregulation and imbalance between the striosome and matrix compartments in the striatum

Autism is characterised by atypical social communication and stereotyped behaviours. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability (ID), but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterised the behaviour of Shank3{Delta}11/{Delta}11 mice from three to twelve months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEG). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g. Grm2, Dlgap1), G-protein-signalling pathways (e.g. Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g. Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal1, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3{Delta}11/{Delta}11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of SHANK3-deficient mice and strongly suggest, for the first time, that the impairment in behaviour of these mice are related to an imbalance striosomes/matrix.

neuroscience↗

bioRxiv2021

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1 activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CDK1 activating kinases (CAK) or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a tripartite complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

biochemistry↗

bioRxiv2021

FISH-quant v2: a scalable and modular analysis tool for smFISH image analysis

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in-situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling a quantitative understanding of gene expression and its regulatory mechanisms. Recent progress in experimental techniques provides larger data-sets, requiring adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular analysis tool accessible both for non-experts and experts, which we validated and applied on large-scale smFISH image datasets. Our package allows the user to detect isolated and clustered mRNA spots, segment nuclei and cells, quantify RNA localization patterns and visualize these results at the single-cell level.

bioinformatics↗

bioRxiv2021

Combinatorial small molecule-mediated activation of CBP/p300 with environmental enrichment in chronic severe experimental spinal cord injury to enable axon regeneration and sprouting for functional recovery

The interruption of spinal circuitry following spinal cord injury disrupts neural activity and is followed by a failure to mount an effective regenerative gene expression response resulting in permanent neurological disability. Functional recovery requires the enhancement of axonal and synaptic plasticity of spared as well as injured fibres, which need to sprout and/or regenerate to form new connections. Here we will investigate whether the combined epigenetic stimulation of the regenerative gene expression program with neuronal activity-dependent enhancement of neuroplasticity and guidance can overcome the current inability to promote neurological recovery in severe and chronic spinal cord injury. This will be carried out by delivering the small molecule CBP/p300 activator CSP-TTK21 in combination with environmental enrichment. We will deliver the CBP/p300 activator CSP-TTK21 weekly between week twelve and twenty following a severe transection model of spinal cord injury in the mouse. Data analysis will assess modifications in regenerative signalling, in motor and sensory axon sprouting and regeneration, in synaptic plasticity as well as in neurological sensorimotor recovery. This approach will advance our mechanistic understanding of regenerative failure and provide insight of whether combining growth promoting strategies with enhancement of neural activity is an effective therapeutic strategy in chronic spinal cord injury.

neuroscience↗

bioRxiv2021

RNA labelling in live plants reveals single cell transcriptional dynamics: application to phosphate signaling

Plants are sessile organisms constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, real time and changing environmental conditions. Using phosphate responsive genes as model, we found that active genes displayed high transcription initiation rates ([~]3s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady-state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpected fast kinetics ([~]minutes) and striking heterogeneity between neighboring cells. Access to single cell RNA polymerase II dynamics within live plants will benefit future studies of signaling processes.

plant biology↗

bioRxiv2021

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.

microbiology↗

bioRxiv2021

Hydroxyapatite pellets as versatile model surfaces for systematic studies on enamel

Research into materials for medical application draws inspiration from naturally occurring or synthesized surfaces, just like many other research directions. For medical application of materials, particular attention has to be paid to biocompatibility, osseointegration and bacterial adhesion behavior. To understand their properties and behavior, experimental studies with natural materials such as teeth are strongly required. The results, however, may be highly case-dependent because natural surfaces have the disadvantage of being subject to wide variations, for instance in their chemical composition, structure, morphology, roughness, and porosity. A synthetic surface which mimics enamel in its performance with respect to bacterial adhesion and biocompatibility would, therefore, facilitate systematic studies much better. In this study, we discuss the possibility of using hydroxyapatite (HAp) pellets to simulate the surfaces of teeth and show the possibility and limitations of using a model surface. We performed single-cell force spectroscopy with single Staphylococcus aureus cells to measure adhesion-related parameters such as adhesion force and rupture length of adhesins binding to HAp and enamel. We also examine the influence of blood plasma and saliva on the adhesion properties of S. aureus. The results of these measurements are matched to water wettability, elemental composition of the samples and the change in the macromolecules adsorbed over time. We found that the adhesion properties of S. aureus were similar on both samples under all conditions: Significant decreases in adhesion strength were found equally in the presence of saliva or blood plasma on both surfaces. We therefore conclude that HAp pellets are a good alternative for natural dental material. This is especially true when slight variations in the physicochemical properties of the natural materials may affect the experimental series.

biophysics↗

bioRxiv2020

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly showed single mRNAs transported by single KIF1C motors, with the 3UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal an RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role both in transporting RNAs and in promoting their clustering within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

cell biology↗

bioRxiv2020

A lightweight segmentation and lineage tracking tool for noisy, low frame rate 4D fluorescence microscopy data

4D fluorescence microscopy allows the study of spatiotemporal cell dynamics in embryonic development in unprecedented detail, yet the uneven scattering of light within developing embryos presents challenges in discerning fine details. We present a tool which pre-processes large in vivo 4D microscopy datasets and can then track the movements and lineage histories of rapidly dividing embryonic stem cells. This solution offers a robust, simple segmentation technique to segment high intensity fluorescent features in highly scattered datasets, such as from deep within a developing zebrafish embryo. This tool then offers lineage tracing functionality by tracking rapidly dividing nuclei and their progeny, while also accounting for the jumps rapidly moving features make between frames in low frame rate data. Based on the user having prior knowledge of their imaging subject, this tool has applications in datasets with limitations with signal to noise, and frame rate. It can provide information regarding the position and movement of rapidly dividing nuclei. We demonstrate this pipeline in developing early zebrafish embryos.

bioinformatics↗

bioRxiv2020

Global regulatory transitions at core promoters demarcate the mammalian germline cycle

Core promoters integrate regulatory inputs of genes1-3. Global dynamics of promoter usage can reveal systemic changes in how genomic sequence is interpreted by the cell4 Here we report the first analysis of promoter dynamics and code switching in the mammalian germ line, characterising the full cycle of transitions from embryonic stem cells through germline, oogenesis, and zygotic genome activation. Using Super Low Input Carrier-CAGE5,6 (SLIC-CAGE) we show that mouse germline development starts with the somatic promoter code, followed by a prominent switch to the maternal code during follicular oogenesis. The sequence features underlying the shift from somatic to maternal code are conserved across vertebrates, despite large differences in promoter nucleotide compositions. In addition, we show that, prior to this major shift, the promoters of gonadal germ cells diverge from the canonical somatic transcription initiation. This divergence is distinct from the promoter code used later by developing oocytes and reveals genome-wide promoter remodelling associated with alternative nucleosome positioning during early female and male germline development. Collectively, our findings establish promoter-level regulatory transitions as a central, conserved feature of the vertebrate life cycle.

developmental biology↗

bioRxiv2020

Chromoplast differentiation in bell pepper (Capsicum annuum) fruits

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell-pepper fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b6f complex, the ATPase complex as well as Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids such as the oxidative pentose phosphate pathway (OPPP), amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins declines but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation may be consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper, that lack a respiratory burst at the onset of fruit ripening.

plant biology↗

bioRxiv2020

Single Cell Transcriptomics of Human Native Kidney and Kidney Allografts and New Insights into the Mechanism of Fibrosis

We tested the hypothesis that single-cell RNA-sequencing (scRNA-seq) analysis of human kidney allograft biopsies will reveal distinct cell types and states and yield insights to decipher the complex heterogeneity of alloimmune injury. We selected 3 biopsies of kidney cortex from 3 individuals for scRNA-seq and processed them fresh using an identical protocol on the 10x Chromium platform; (i) HK: native kidney biopsy from a living donor, (ii) AK1: allograft kidney with transplant glomerulopathy, tubulointerstitial fibrosis, and worsening graft function, and (iii) AK2: allograft kidney after successful treatment of active antibody-mediated rejection. We did not study T-cell-mediated rejections. We generated 7217 high-quality single cell transcriptomes. Taking advantage of the recipient-donor sex mismatches revealed by X and Y chromosome autosomal gene expression, we determined that in AK1 with fibrosis, 42 months after transplantation, more than half of the kidney allograft fibroblasts were recipient-derived and therefore likely migratory and graft infiltrative, whereas in AK2 without fibrosis, 84 months after transplantation, most fibroblasts were donor-organ-derived. Furthermore, AK1 was enriched for tubular progenitor cells overexpressing profibrotic extracellular matrix genes. AK2, eight months after successful treatment of rejection, contained endothelial cells that expressed T-cell chemoattractant cytokines. In addition to these key findings, our analysis revealed unique cell types and states in the kidney. Altogether, single-cell transcriptomics yielded novel mechanistic insights, which could pave the way for individualizing the care of transplant recipients.

molecular biology↗

bioRxiv2020

A systematic genetic interaction map of human solute carriers assigns a role to SLC25A51/MCART1 in mitochondrial NAD uptake

Solute Carriers (SLCs) represent the largest family of human transporter proteins, consisting of more than 400 members1,2. Despite the importance of these proteins in determining metabolic states and adaptation to environmental changes, a large proportion of them is still orphan and lacks associated substrates1,3,4. Here we describe a systematic mapping of genetic interactions among SLCs in human cells. Network-based identification of correlated genetic interaction profile neighborhoods resulted in initial functional assignments to dozens of previously uncharacterized SLCs. Focused validation identified SLC25A51/MCART1 as the SLC enabling mitochondrial import of NAD(H). This functional interaction map of the human transportome offers a route for systematic integration of transporter function with metabolism and provides a blueprint for elucidation of the dark genome by biochemical and functional categories.

cell biology↗

bioRxiv2020

TBPL2/TFIIA complex overhauls oocyte transcriptome during oocyte growth

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

molecular biology↗

bioRxiv2020

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37{degrees}C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4{degrees}C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

neuroscience↗

bioRxiv2020

Clustering and reverse transcription of HIV-1 genomes in nuclear niches of macrophages

In order to replicate, the Human Immunodeficiency Virus (HIV-1) reverse transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early replication cycle in macrophages, natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA and viral RNA, in which multiple genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6 and localize to nuclear speckles. Strikingly, we show that viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where RT is pharmacologically suppressed and in untreated cells. We demonstrate that, after temporary inhibition, RT can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear RT can result in transcription competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle, and may have implications for understanding HIV-1 persistence.

microbiology↗