Biology subjects

Sachs, M.

Publications and source records attributed to Sachs, M..

6 recordsLinked to original sources

bioRxiv2025

The morphology and small subunit rDNA gene phylogeny of the novel goniomonad genus Ebisugoniomonas and two novel Poseidogoniomonas species

Goniomonads are heterotrophic representatives of Cryptista, sister lineage to the phototrophic Cryptists. Goniomonads are morphologically and genetically diverse, comprising nine genera described so far. Besides morphologically described genera, SSU rDNA sequencing data has revealed the existence of at least one more monophyletic genus-level lineage within goniomonads, as well as more novel species within the described genera. Furthermore, the diversity of SSU rDNA sequences within goniomonads is not fully investigated, and addition of novel sequences can be crucial for understanding the evolution and diversity of goniomonads and for providing a basis for taxonomic descriptions. The morphological data record available for goniomonads is relatively limited: many cellular features remain undescribed, or described only for a few isolates. In this study, we describe the new genus and species Ebisugoniomonas hispanica n. gen. n. sp., and two novel species within Poseidogoniomonas: P. posteriocircus n. sp. and P. zopfkaesiformis n. sp. We report a range of observations on the cellular features of goniomonads: periplast plate arrangement pattern and intracellular structures. We also update the SSU rDNA phylogeny of goniomonads by adding sequences from novel isolates and environmental DNA studies. Several sequences recovered from novel isolates and environmental DNA datasets likely represent highly divergent lineages of goniomonads in the SSU rDNA phylogeny.

microbiology↗

bioRxiv2025

Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue in vitro maturation enhances maturation rates and modulates oocyte proteome and ultrastructure

Study questionCould follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue in vitro maturation (rIVM)? Summary answerSupplementation of rIVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by >20%, inducing changes in oocyte protein profile and organelle distribution. What is already knownIVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could enhance oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, durationWomen undergoing transvaginal oocyte retrieval after COS (n=83) were recruited to donate follicular fluid (n=54 single follicles) and/or immature GV oocytes (n=95). We aimed to: a) define differences in the protein cargo of ffEVs derived from human follicles containing mature (MII-ffEVs, n=10) versus immature (GV-ffEVs, n=5; metaphase I MI-ffEVs, n=5) oocytes, b) demonstrate the capacity of human GV oocytes to uptake MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methodsffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n=15) was assessed by confocal microscopy. GVs were cultured for rIVM in a timelapse incubator with MII-ffEVs (n=45 GVs) or without (n=40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance on transmission electron microscopy (TEM). Main results and the role of chanceWe identified 1340 proteins in ffEVs, with proteins such as F12, IGKV1-39, FREM2, and C1QC being significantly enriched in MII-ffEVs. GV oocytes internalised MII-ffEVs, and their supplementation for 48 hours increased the oocyte maturation rate compared to control by 22.8{+/-}9.4% (77.8% vs 55% maturation rate respectively; p-value=0.0372). Proteomic analysis of ffEV-supplemented mature oocytes (n=6) revealed 56 differentially abundant proteins (DAPs) compared to not supplemented mature oocytes (n=5). Among them, 37 DAPs were in higher abundance in ffEVs- supplemented mature oocytes including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). Electron microscopy showed differences in oocyte organelle distribution and appearance, particularly that of endoplasmic reticulum (RE) and RE-mitochondria complexes. Functional enrichment analysis of differentially abundant proteins during ffEV-oocyte interaction revealed regulation of endoplasmic reticulum, steroid biosynthesis, and keratin organisation pathways. Large scale dataN/A Limitations, reasons for cautionThis study utilised immature oocytes from COS cycles, therefore the results should be interpreted within the context of rIVM potential. Winder implications of the findingsThese results provide new insights into the role of ffEVs in enhancing oocyte maturation, offering potential improvements for clinical rIVM protocols and inspire the development of global IVM supplements based on ffEVs or associated specific cargo. Study funding/competing interest(s)This work was funded by an EMDO research fellowship and a FAN research grant (Fonds zur Forderung des akademischen Nachwuchses) from the University of Zurich. What does it mean for the patientsInfertility rates are rising, with 17% of couples worldwide needing help to get pregnant, often through treatments like in vitro fertilisation (IVF). IVF usually involves using hormones to stimulate the ovaries to produce multiple eggs, which can be tough on a womans health, both physically and emotionally, and can be very expensive. In vitro oocyte maturation (IVM) is a gentler alternative, where eggs are matured outside the body, reducing risks and costs. However, IVM isnt as effective as IVF yet, mainly because the current methods are not perfect. Our research is exploring a new approach to improve IVM by adding extracellular vesicles from follicular fluid to the egg culture. This could help the eggs mature better, leading to higher success rates and giving more options to couples struggling with infertility.

cell biology↗

bioRxiv2024

Essential genes encoded by the mating-type locus of the human fungal pathogen Cryptococcus neoformans

Fungal sexual reproduction is controlled by the mating-type (MAT) locus. In contrast to a majority of species in the phylum Basidiomycota that have tetrapolar mating-type systems, the opportunistic human pathogen Cryptococcus neoformans employs a bipolar mating-type system, with two mating types (a and ) determined by a single MAT locus that is unusually large ([~]120 kb) and contains more than 20 genes. While several MAT genes are associated with mating and sexual development, others control conserved cellular processes (e.g. cargo transport and protein synthesis), of which five (MYO2, PRT1, RPL22, RPL39, and RPO41) have been hypothesized to be essential. In this study, through genetic analysis involving sporulation of heterozygous diploid deletion mutants, as well as in some cases construction and analyses of conditional expression alleles of these genes, we confirmed that with the exception of MYO2, both alleles of the other four MAT genes are indeed essential for cell viability. We further showed that while MYO2 is not essential, its function is critical for infectious spore production, faithful cytokinesis, adaptation for growth at high temperature, and pathogenicity in vivo. Our results demonstrate the presence of essential genes in the MAT locus that are divergent between cells of opposite mating types. We discuss possible mechanisms to maintain functional alleles of these essential genes in a rapidly-evolving genomic region in the context of fungal sexual reproduction and mating-type evolution. IMPORTANCESexual reproduction is essential for long-term evolutionary success. Fungal cell type identity is governed by the MAT locus, which is typically rapidly evolving and highly divergent between different mating types. In this study, we show that the a and alleles of four genes encoded in the MAT locus of the opportunistic human fungal pathogen C. neoformans are essential. We demonstrate that a fifth gene, MYO2, which had been predicted to be essential, is in fact dispensable for cell viability. However, a functional MYO2 allele is important for cytokinesis and fungal pathogenicity. Our study highlights the need for careful genetic analyses in determining essential genes, which is complementary to high-throughput approaches. Additionally, the presence of essential genes in the MAT locus of C. neoformans provides insights into the function, maintenance, and evolution of these fast-evolving genomic regions.

genetics↗

bioRxiv2024

Cryptic Cryptophytes-revision of the genus Goniomonas

Cryptomonad protists are ubiquitously distributed over marine and freshwater habitats. As an exception to the coloured cryptomonads, the heterotrophic cryptomonads of the genus Goniomonas have an ancestral phylogenetic position. They lack any kind of chloroplast and most likely represent a basal group to those cryptomonad groups having obtained their chloroplast by secondary endosymbiosis. Earlier studies have shown a deep divergence between freshwater and marine clades of goniomonads that comprise large genetic distances between members within the group and also between the two groups of marine and freshwater taxa. Still, marine and freshwater species carry the same genus name, and until up to date only few species have been described. We therefore restructured goniomonad systematics based not only on a separation of marine and freshwater taxa, but, taking the large genetic distances into account, also several new genera are described. Based on morphological as well as phylogenetic data (18S rDNA sequences), this leads to the formation of the freshwater genera Limnogoniomonas n. g., Goniomonas and Aquagoniomonas n. g. and the marine genera Neptunogoniomonas n. g., Baltigoniomonas n. g., Marigoniomonas n. g., Thalassogoniomonas n. g., Poseidogoniomonas n. g. and Cosmogoniomonas n. g. To give the restructuring process a stable basis, we additionally propose a neotype for Goniomonas truncata.

molecular biology↗

bioRxiv2024

Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/beta-catenin signaling

Activation of the Wnt/{beta}-catenin pathway crucially depends on polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/{beta}-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.

cell biology↗

bioRxiv2020

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

PurposeThe kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). MethodsGlomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis. ResultsTimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli. ConclusionGlobally applicable timMEP abolishes the need for costly, time- and animal-consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology. Key messagesO_LIA tripartite isolation method for mesangial, endothelial and podocyte cell-types from reporter-free mice. C_LIO_LIGeneration of bulk cell-type samples and primary co-cultures for biochemical and protein-based analyses. C_LIO_LIStrain and transgene-dependent expression of proteins among glomerular cell-types, including protein profiles, intra-glomerular communication machineries, and reporter-dependent artefacts. C_LIO_LIDisease specific time-resolved resolution of glomerular cell-types response to injury. C_LI

cell biology↗