Biology subjects

Zimmer, C.

Publications and source records attributed to Zimmer, C..

32 records · Page 2Linked to original sources

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

FKBP5 expression is related to HPA flexibility and the capacity to cope with stressors in the house sparrow

The hypothalamic-pituitary-adrenal (HPA) axis and its end products, the glucocorticoids, are critical to responding appropriately to stressors. Subsequently, many studies have sought relationships between glucocorticoids and measures of health or fitness, but such relationships are at best highly context dependent. Recently, some endocrinologists have started to suggest that a focus on HPA flexibility, the ability of an individual to mount appropriate responses to different stressors, could be useful. Here, we tested the hypothesis that expression of FKBP5, a cochaperone in the glucocorticoid receptor complex, is a simple and reliable proxy of HPA flexibility in a wild songbird, the house sparrow (Passer domesticus). We quantified HPA flexibility in a novel way, using guidance from research on heart rhythm regulation. As predicted, we found that adult sparrows with low stress-induced FKBP5 expression in the hypothalamus exhibited high HPA flexibility. Moreover, low FKBP5 expression was associated with better stress coping capacities in terms of exploratory disposition and body mass maintenance. Altogether, these results suggest that FKBP5 may be important in the regulation HPA flexibility, potentially affecting how individuals cope with natural and anthropogenic adversity.

physiology↗

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↗

bioRxiv2020

Analysis of the intrinsic chromatin binding property of HIV-1 integrase and its regulation by LEDGF/p75 using human chromosomes spreads

Retroviral integration requires the stable insertion of the viral genome into the host chromosomes. During this process, the functional integration complex must associate with cellular chromatin via the interaction between retroviral integrase and nucleosomes. The final association between the HIV-1 integration complex and the nucleosomal target DNA remains unclear and may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To date, there is no experimental system allowing the direct monitoring of this protein association with chromatin to depict the molecular mechanism of this process fully. To investigate this and understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase further, we used both biochemical approaches and an unedited chromosome-binding assays. Our study revealed that retroviral IN has an intrinsic ability to bind and recognize specific chromatin regions even in the absence of its cofactor. We also showed that this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromatin regions. Using these approaches, we also better determined the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. In addition to supporting a chromatin-binding function of the integrase protein acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate, our work precisely elucidates the mechanism of action of LEDGF/p75 in this crucial integration step.

microbiology↗

bioRxiv2020

Migraine Attacks as a Result of Hypothalamic Loss of Control

To assess the natural trajectory of brain activity over the migraine cycle, we assessed (1) the cerebral perfusion and (2) the hypothalamic connectivity during spontaneous headache attacks with follow-up recordings towards the next migraine attack. Using pseudo-continuous arterial spin labelling and resting-state functional magnetic resonance imaging, 12 migraine patients were examined in 82 sessions. We detected cyclic changes of brain perfusion in the limbic circuit (insula, hippocampus, and nucleus accumbens), with the highest perfusion during headache attacks. In addition, we found an increase of hypothalamic connectivity to the limbic system over the interictal interval, then collapsing during the headache. Our data provide strong evidence for the predominant role of the hypothalamus as a zeitgeber for generating migraine attacks. Our findings suggest that migraine attacks are the result of the hypothalamus losing control over the limbic system.

neuroscience↗

bioRxiv2020

A localization screen reveals translation factories and widespread co-translational RNA targeting

Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For {beta}-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

cell biology↗

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↗

bioRxiv2020

Improving the chromosome-level genome assembly of the Siamese fighting fish (Betta splendens) in a university Master's course.

BackgroundEver decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university Masters course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behaviour. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published HiC data. FindingsThe use of nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using previously published HiC data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 95.8% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. ConclusionWe present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university Masters course. The use of ~35x long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

evolutionary biology↗

bioRxiv2019

Chromosome-level quality scaffolding of brown algal genomes using InstaGRAAL, a proximity ligation-based scaffolder

Hi-C has become a popular technique in recent genome assembly projects. Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs in draft genomes, resulting in chromosome-level assemblies. However, application of this approach is currently hampered by a lack of robust programs that are capable of effectively treating this type of data, particularly open source programs. We developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. Both GRAAL, and instaGRAAL use a Markov Chain Monte Carlo algorithm to perform Hi-C scaffolding, but instaGRAAL features a number of improvements including a modular polishing approach that optionally integrates independent data. To validate the program, we used it to generate chromosome-level assemblies for two brown algae, Desmarestia herbacea and the model Ectocarpus sp., and quantified improvements compared to the initial draft for the latter. Overall, instaGRAAL is a program able to generate, using default parameters with minimal human intervention, near-complete assemblies.

genomics↗

bioRxiv2019

Environmental unpredictability shapes glucocorticoid regulation across populations of tree swallows

Responding appropriately to challenges is an important contributor to fitness. Variation in the regulation of glucocorticoid hormones, which mediate the phenotypic response to challenges, can therefore influence the ability to persist in a given environment. We compared stress responsiveness in four populations of tree swallows (Tachycineta bicolor) along an environmental gradient to evaluate support for different selective pressures in driving the evolution of glucocorticoid regulation. In accordance with the environmental unpredictability hypothesis, stronger stress responses were seen in more unpredictable environments. Contrary to the reproductive value hypothesis, the stress response was not lower in populations engaging in more valuable reproductive attempts. Populations with stronger stress responses also had stronger negative feedback, which supports a "mitigating" rather than a "magnifying" effect of negative feedback on stress responses. These results suggest that combining a robust stress response with strong negative feedback may be important for persisting in unpredictable or rapidly changing environments.

evolutionary biology↗

bioRxiv2019

Plumage manipulation alters the integration of social behavior, physiology, internal microbiome, and fitness.

Signals often covary with physiological and behavioral traits to form an axis of integrated phenotypic variation associated with reproductive performance. This pattern of phenotypic integration could result from intrinsic between-individual differences that are causally related to signal production, physiology, and behavior. Alternatively, signal expression itself might generate dynamic feedback between physiology, behavior, and the experienced social environment, resulting in an integrated phenotype. Here, we manipulated the plumage of female tree swallows (Tachycineta bicolor) to decouple the expression of a social signal from any pre-existing behavioral or physiological differences. We collected a time series of physiological samples, monitored social interactions with a sensor network, and tracked reproductive performance. Relative to sham controls, dulled females experienced an altered social environment; overall, these females were visited more by conspecific females and less by males. Dulled females subsequently changed their own behavior by initiating fewer interactions and increasing nestling provisioning. These differences resulted in an altered internal microbiome and glucose levels and, ultimately, dulled females produced more offspring. Moreover, dulled females produced larger clutches than control females in the year after the manipulation. Thus, signal variation alone--independent from any pre-existing differences--had a sustained causal affect on a suite of integrated traits. This finding suggests that dynamic feedback may play an important role in coordinating an integrated signaling phenotype. Our results have implications for understanding how variation in signal expression arises and is maintained and the extent to which the information encoded in signals is contingent upon their use in a social environment.

zoology↗

bioRxiv2019

Human subsystems of medial temporal lobes extend locally to amygdala nuclei and globally to an allostatic-interoceptive system

In mammals, the hippocampus, entorhinal, perirhinal, and parahippocampal cortices (i.e., core regions of the human medial temporal lobes, MTL) are locally interlaced with the adjacent amygdala nuclei at the structural and functional levels. At the global brain level, the human MTL has been described as part of the default mode network whereas amygdala nuclei as parts of the salience network, with both networks forming collectively a large-scale brain system supporting allostatic-interoceptive functions. We hypothesized (i) that intrinsic functional connectivity of slow activity fluctuations would reveal human MTL subsystems locally extending to the amygdala; and (ii) that these extended local subsystems would be globally embedded in large-scale brain systems supporting allostatic-interoceptive functions. From the resting-state fMRI data of three independent samples of cognitively healthy adults (one main and two replication samples: Ns = 101, 61, and 29, respectively), we analyzed the functional connectivity of fluctuating ongoing BOLD-activity within and outside the amygdala-MTL in a data-driven way using masked independent component and dual-regression analyses. We found that at the local level MTL subsystems extend to the amygdala and are functionally organized along the longitudinal amygdala-MTL axis. These subsystems were characterized by a consistent involvement of amygdala, hippocampus, and entorhinal cortex, but a variable participation of perirhinal and parahippocampal regions. At the global level, amygdala-MTL subsystems selectively connected to salience, thalamic-brainstem, and default mode networks - the major cortical and subcortical parts of the allostatic-interoceptive system. These results provide evidence for integrated amygdala-MTL subsystems in humans, which are embedded within a larger allostatic-interoceptive system.

neuroscience↗

bioRxiv2019

Does variation in glucocorticoid regulation predict fitness? A phylogenetic meta-analysis.

Glucocorticoid hormones (GCs) are central mediators of metabolism and the response to challenges. Because circulating levels of GCs increase in response to challenges, within-population variation in GCs could reflect individual variation in condition or experience. At the same time, individual variation in the degree to which GCs increase in response to challenges (which is relatively consistent within individuals over time) could have causal effects on stress coping capacity, and the ability to survive and reproduce. Although a number of studies in vertebrates have tested whether within-population variation in GCs predicts components of fitness, it is not clear whether there are consistent patterns across taxa. Here we present the first phylogenetic meta-analysis testing whether within-population variation in GCs is associated with components of fitness across vertebrates. At the same time, we introduce and test predictions about an overlooked but potentially important mediator of GC-fitness relationships: life history context. We suggest that strong context-dependence in the fitness benefit of maintaining elevated GCs could obscure consistent patterns between GCs and fitness across taxa. Meta-analyses revealed that across vertebrates, baseline and stress-induced GCs were consistently negatively correlated with reproductive success. This relationship did not differ depending on life history context. In contrast, the relationships between GCs and survival were highly context dependent, differing according to life history strategy. Both baseline and stress-induced GCs were more strongly negatively associated with survival in longer-lived populations and species. Stress-induced GCs were also more negatively associated with survival in organisms that engage in relatively more valuable reproductive attempts. Fecal GCs did not predict survival or reproductive success. We also used a meta-analytic approach to test whether experimental increases in GCs had consistent causal effects on fitness. Experimental increases in GCs reduced both survival and reproductive success, although the latter relationship was not significant when accounting for phylogeny. Overall, these results support the prediction that GC-fitness relationships can be strongly context dependent, and suggest that incorporating life history may be particularly important for understanding GC-survival relationships. Future studies that explore the role of other aspects of context (e.g., the nature and frequency of stressors, environmental variation) within and across species could provide important insights how and when variation in GC regulation predicts fitness.

evolutionary biology↗

bioRxiv2017

A Versatile Compressed Sensing Scheme For Faster And Less Phototoxic 3D Fluorescence Microscopy

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.\n\nOCIS codes: (110.1758) Computational imaging; (170.2520) Fluorescence microscopy; (170.6900) Three-dimensional microscopy.

biophysics↗