Biology subjects

Frey, C.

Publications and source records attributed to Frey, C..

8 recordsLinked to original sources

bioRxiv2026

Fluorescence based microviscosity mapping in membraneless organelles

Membraneless organelles (MLOs) are cellular biomolecular condensates formed by liquid-liquid phase separation. Their biological functions are intimately linked to their material properties including viscosity. Condensate viscosity is determined by the size, shape, concentration and molecular interactions between MLOs components. It impacts the diffusion of MLOs constituents and the selective permeability of the condensate, thereby regulating the rate of biochemical reactions. Viscosity modifications associated with liquid-to-gel transition of the condensates are related to pathologies. Current experimental approaches for characterizing the material properties of cellular condensates remain limited. In this study, we report the use of BODIPY-based molecular rotors, in combination with fluorescence lifetime imaging microscopy (FLIM), to monitor the microviscosity of cellular MLOs directly in living cells. The fluorescence lifetime of BODIPY derivatives increases with the viscosity of their microenvironment, enabling quantitative assessment of microviscosity within condensates. HaloTag technology was employed to specifically label MLO components. Our findings reveal that the nucleolus exhibits higher viscosity than the surrounding nucleoplasm and that microviscosity varies across nucleolar sub-compartments. Furthermore, nucleolar reorganization induced by inhibition of rRNA synthesis results in a measurable increase in microviscosity. Finally, we demonstrate that the microviscosity of stress granules is lower than that of the nucleolus. Overall, presented results demonstrate the strong potential of the BODIPY based molecular rotors as a versatile and powerful tools for probing the material properties of cellular MLOs.

biophysics↗

bioRxiv2025

Non-canonical reductive nitrous oxide production pathways in a seasonally stratified lake basin

Nitrous oxide (N2O), a potent greenhouse gas and ozone-depleting agent, was detected at high concentrations in the anoxic bottom-waters of a monomictic eutrophic lake basin in Switzerland (Lake Lugano). The observed high site-specific nitrogen (N) isotope preference (SP), was inconsistent with bacterial denitrification, which typically exhibits low SP, thereby challenging its role as the primary N2O source. This pointed to chemo-denitrification and/or fungal denitrification, both characterized by high SP, as possible alternative pathways. We conducted incubation experiments with sediment and bottom-water samples to assess N2O production and reduction dynamics and associated natural-abundance stable isotope signatures. We demonstrate that N2O accumulation predominantly originated from sedimentary production, and that elevated SP values in the bottom water reflected fractional bacterial N2O reduction. Using an isotope mass balance mixing model, we identified bacterial denitrification as the dominant sedimentary process ([~]75%), followed by chemo-denitrification ([~]20%), and fungal denitrification ([~]5%). Additional 15N tracer incubation experiments, combined with selective inhibitors to quantify isolated pathways, confirmed model-estimated contributions. These findings validate the use of literature-based SP values in mixing models, and provide evidence for non-canonical N2O production via chemo- and fungal denitrification, highlighting the need to broaden our understanding of N2O cycling in lakes beyond classical bacterial pathways. SYNOPSISEutrophic lake conditions significantly enhance nitrous oxide production through diverse microbial processes, including non-traditional pathways beyond classical nitrification and denitrification. This study highlights the importance of incorporating chemo- and fungal denitrification when investigating aquatic nitrous oxide cycling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/670496v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@808806org.highwire.dtl.DTLVardef@626820org.highwire.dtl.DTLVardef@a22bd8org.highwire.dtl.DTLVardef@eca378_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Long-term euxinia hinders microbial ammonium removal in brackish coastal waters

Anthropogenic activities are key drivers of eutrophication and deoxygenation in coastal marine ecosystems. This stimulates the anaerobic degradation of organic matter and the release of reduced products, such as ammonium, methane, and hydrogen sulfide, which may, in turn, exacerbate eutrophication and deoxygenation. In this study, using a combination of chemical and microbial analyses, we assess the nitrogen dynamics in the water column of a eutrophic coastal system (Stockholm Archipelago) at three sites with contrasting redox conditions (oxic to long-term euxinic). At the oxic site, counter gradients of ammonium and oxygen in the water column, low nitrate {delta}15N values in bottom waters, and the 16S rRNA gene-based presence of nitrifiers indicate nitrification near the sediment-water interface. At the seasonally and long-term euxinic sites, nitrification, as inferred from the water column oxygen and nutrient profiles and the relative abundance of nitrifiers, primarily occurred near the oxycline. At these two sites, nitrate was removed below the oxycline through denitrification linked to sulfide oxidation by Sulfurimonas. Nitrous oxide emissions from surface waters in the archipelago reached up to 40 {micro}mol m-2 d-1 and were not directly related to water column redox conditions, indicating that multiple factors control coastal emissions of this greenhouse gas to the atmosphere. The relative abundance of 16S rRNA genes and of N-cycle genes in metagenomes was highest at the seasonally euxinic site. Importantly, nitrifiers were significantly less abundant at the long-term euxinic site. Our results highlight that prolonged euxinia promotes recycling of ammonium over its removal, likely due to sulfide inhibition of nitrification, which sustains eutrophication and deoxygenation of coastal systems.

microbiology↗

bioRxiv2025

Nitrogen and oxygen isotope effects during enzymatic nitrate reduction in vitro and by natural lake water consortia

Nitrate (NO3-) isotope ratios are useful indicators for nitrogen (N)-transformation processes if the associated isotope effects and their environmental controls are well-constrained. The NO3- isotope effects in natural environments may depend on the type of dissimilatory nitrate reductases involved and the degree of isotopic overprinting via NO3- regeneration. We measured the coupled N and oxygen (O) isotope effects of NO3- reduction in anoxic incubation experiments with laboratory cultures (Pseudomonas sp. and Escherichia coli) harboring different nitrate reductase enzymes (Nar and/or Nap) as well as with natural freshwater consortia from Lake Lugano (Switzerland) and Lake La Cruz (Spain). For comparison, isotope effects were also evaluated through coupled N and O isotope measurements in the redox transition zone of Lake Lugano North Basin. Incubation-based Rayleigh model N isotope effects ({varepsilon}N) were variable, ranging from 9 to 30{per thousand}. In comparison, in situ {varepsilon}N values (5 to 14{per thousand}) estimated by the closed system model were lower, likely due to substrate limitation in the water column. Experiments with Pseudomonas sp. and E. coli cultures possessing Nar yielded N isotope effects of similar magnitudes and, consistent with previous data, robust {Delta}{delta}18O:{Delta}{delta}15N enrichment ratios of [~]0.9 - 1.0. Nitrate reduction by cultures possessing solely Nap led to lower {Delta}{delta}18O:{Delta}{delta}15N of [~]0.7. In anoxic incubations of lake water, where the effect of nitrification could be excluded, {Delta}{delta}18O:{Delta}{delta}15N values between 0.6 and 1.0 suggest "community activity" of both Nar and Nap. Interestingly, stimulation of lithotrophic nitrate reduction in incubations with amended NO3- + sulfide resulted in a {Delta}{delta}18O:{Delta}{delta}15N slope of 0.90 {+/-} 0.03 (standard error, SE), indicating Nar as the more dominant nitrate-reducing enzyme. On the contrary, stimulation of organotrophic nitrate reduction in NO3- + acetate amended incubations resulted in a significantly lower slope of 0.72 {+/-} 0.03 SE, suggesting a greater contribution by Nap. In contrast to the nitrate-reduction incubation experiments, the in situ {Delta}{delta}18O:{Delta}{delta}15N value of 1.36 {+/-} 0.14 SE observed in the Lake Lugano water column was unusually high for a freshwater environment, likely reflecting the superimposed effect of NO3- production by nitrification. Our study thus underscores that both variations in activity of Nar versus Nap during nitrate reduction, as well as isotopic overprinting by nitrate regeneration may impact ecosystem NO3- isotope dynamics in natural denitrifying environments.

microbiology↗

bioRxiv2025

Comparative spatial transcriptomics of hair follicle-T cell interactions in mouse, dog and human reveals conserved drivers of primary cicatricial alopecia

Primary cicatricial alopecias (PCA) encompass several autoimmune disorders characterized by scarring hair loss. Many of these conditions are lymphocytic and are thought to be driven by T cell populations. Here, we sought to characterize potential T cell-hair follicle communication pathways in the microanatomical niche using spatial transcriptomics across 3 mammalian species including a novel mouse model, spontaneous disease in companion dogs and human archival diagnostic biopsies. Flow cytometry of mouse model skin confirmed loss of CD34+ bulge cells and keratinocytes, and bulk microarray and histology revealed expression of collagens and development of fibrosis. In vivo ear imaging in mice engrafted with Kikume photoconvertible OT1 CD8+ T cells confirmed long-lived RFP+ T cells in skin arrest near hair follicles and recruit other GFP+ T cells. OT1 T cells expressed CD69, CD103, CD122 and CD62L, which is a binding partner of CD34. Digital spatial profiling using CD3, CD8 and CD45 cell masking identified CXCR3 ligands and IFN response genes in hair follicles, and "metabolic" pathways in T cells, which were also recapitulated in dog and human biopsies. Bulk human RNA as well as spatial analysis of perifollicular T cells confirmed enrichment of CD69 and SELL/CD62L. Different pathways predominated in other CD3+ regions of interest in CD4+ driven conditions including mucocutaneous lupus erythematosus and subacute cutaneous lupus erythematosus. Last, we identify novel drug-targetable pathways, namely CFD and S100A8/9, that could be further explored to disrupt processes in these conditions through veterinary and human trials.

immunology↗

bioRxiv2024

Holoprosencephaly and Cyclopia in bmp7b and bmpr1ba Crispant zebrafish

Holoprosencephaly (HPE) is the most frequent developmental disorder of the forebrain. In HPE, the early single anlage of the forebrain, the Anterior Neural Plate (ANP) which encompasses the future telencephalon and eye field, fails to divide. BMP signaling and antagonism are overall important for nervous system development. The focus of this study was on the role of the ligand bmp7b and the receptor bmpr1ba during forebrain development. The zebrafish loci of bmp7b and bmpr1ba were targeted transiently with CRISPR/Ca9. Crispants for both bmp7b and bmpr1ba presented HPE and cyclopia, one central eye. Subsequently, the ANP was addressed in bmp7b Crispants. The morphology of the eye field was affected, with important markers, rx3, six3b and cxcr4a expressed condensed at the midline. Induced expression of bmp4 is also known to result in HPE. Such bmp4 induction altered the expression of bmpr1ba. Zebrafish Crispants for bmp7b and bmpr1ba can be used as a novel HPE model. A challenge in future analyses will be the penetrance of phenotypes in Crispants. Advantages are, however, that analyses can be conducted anywhere, without the need of mutant lines. One important aspect for future analysis will be the role of individual bmp ligands, receptors and antagonists in forebrain development.

developmental biology↗

bioRxiv2024

Reproductive success of inbred strain MV31 of the ctenophore Mnemiopsis leidyi in a self-sustaining inland laboratory culture system

Ctenophores are an attractive lineage for studying animal evolution due to their early divergence from other metazoans. Among Ctenophora, Mnemiopsis leidyi is a model system for developmental, cellular, molecular genetic, and evolutionary studies. Until recently, many of these studies were conducted on wild-caught animals, limiting access to researchers on the coast. Here we present significant advancements towards culturing M. leidyi in laboratories without coastal access, enabling its wider use as an experimental and genetic model system. We detail updated feeding regimes that take advantage of co-culturing Brachionus rotifers with Apocyclops copepods, and quantify the reproductive output of our M. leidyi lab strain on this diet. Our updated feeding regime maintains reproductive fitness comparable to wild-caught individuals. Importantly, we have eliminated the logistical complexities and costs of regularly feeding live larval fish to M. leidyi. Our updated protocols make it feasible to maintain continuous ctenophore cultures independent of access to both coastal populations of wild M. leidyi and larval fish culturing facilities.

zoology↗

bioRxiv2021

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

Bottom-up synthetic biology thrives to reconstruct basic cellular processes into a minimalist cellular replica to foster their investigation in greater details with a reduced number of variables. Among these cellular features, the endomembrane system is an important aspect of cells which is at the origin of many of their functions. Still, the reconstruction of these inner compartments within a lipid-based vesicle remains challenging and poorly controlled. Herein, we report the use of pH as external trigger to self-assemble compartmentalized giant unilamellar vesicles (GUVs) by either bulk, or droplet-based microfluidics. By co-encapsulating pH sensitive small unilamellar vesicles (SUVs), negatively charged SUVs and/or proteins, we show that acidification of the droplets efficiently produces GUVs while sequestrating the co-encapsulated material with flexibility and robustness. The method enables the simultaneous reconstruction of more than a single cellular phenotype from the bottom-up, corresponding to an important advancement in the current status quo of bottom-up synthetic biology.

synthetic biology↗