Biology subjects

Schmidt, A.

Publications and source records attributed to Schmidt, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Integrated data from R405W desmin knock-in mice highlight alterations of mitochondrial function, protein quality control, and myofibrillar structure in the initial stages of myofibrillar myopathy

BackgroundMutations in the desmin gene cause skeletal myopathies and cardiomyopathies. The objective of this study was to elucidate the molecular pathology induced by the expression of R405W mutant desmin in murine skeletal muscle tissue. MethodsA comprehensive characterization of the skeletal muscle pathology in hetero- and homozygous R405W desmin knock-in mice was performed, employing grip strength, blood acylcarnitine and amino acid, histological, ultrastructural, immunofluorescence, immunoblot, ribosomal stalling, RNA sequencing and proteomic analyses. ResultsBoth hetero- and homozygous R405W desmin knock-in mice showed classical myopathological features of a myofibrillar myopathy with desmin-positive protein aggregation, degenerative changes of the myofibrillar apparatus, increased autophagic build-up, and mitochondrial alterations. Muscle weakness and increased blood concentrations of acylcarnitines and amino acids were only present in homozygous animals. During its translation, mutant desmin does not induce terminal ribosomal stalling. Analyses of RNA sequencing and proteomic data from soleus muscle of 3-month-old mice depicted 59 up- and 2 down-regulated mRNAs and 101 up- and 18 down-regulated proteins that were shared between the heterozygous and homozygous genotypes in the respective omics datasets compared to the wild-type genotype. Combined analysis of the omics data demonstrated 187 significantly dysregulated candidates distributed across four groups of regulation. A down-regulation on the mRNA and protein levels was observed for a multitude of mitochondrial proteins including essential proton gradient-dependent carriers. Up-regulation on both omics levels was present for the transcription factor Mlf1, which is a binding partner of protein quality control related Dnajb6. Down-regulated on mRNA but up-regulated on the protein level was the sarcomeric lesion marker Xirp2 (xin actin-binding repeat-containing protein 2), whereas Ces2c (acylcarnitine hydrolase) was regulated in the opposite way. ConclusionsThe present study demonstrates that the expression of mutant desmin results in a myofibrillar myopathy in hetero- and homozygous R405W desmin knock-in mice. Combined morphological, transcriptomic and proteomic analyses helped to decipher the complex pattern of early pathological changes induced by the expression of mutant desmin. Our findings highlight the importance of major mitochondrial alterations, including essential proton gradient-dependent carriers as well as Dnajb6-related protein quality control and Xin-related myofibrillar damage, in the molecular pathogenesis of desminopathies.

neuroscience↗

bioRxiv2024

Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenografts and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

cancer biology↗

bioRxiv2024

The major nucleoid-associated protein WHIRLY1 promotes chloroplast development in barley

WHIRLY1 is a DNA-binding protein of high abundance in chloroplast nucleoids, which have a complex proteome consisting of proteins involved in gene expression and unexpected proteins indicating links to energy production and biosynthetic activities of chloroplasts. In addition, WHIRLY1 has a second localization in the nucleus making it an excellent candidate for chloroplast-to-nucleus communication. To unravel the role of WHIRLY1 for structure and protein composition of nucleoids and its potential involvement in retrograde signaling during chloroplast development, knockout mutants of HvWHIRLY1 were prepared by site-directed mutagenesis using Cas9 endonuclease. In contrast to mutants of rice and maize, which die after the seedling stage, the barley why1 mutants survive and produce grains. Leaves of the mutants are initially pale and get green with time (xantha-to-green phenotype). However, the chlorophyll content of primary leaves stayed distinctly lower than that of the wild-type leaves, coinciding with a rather heterogeneous plastid population, whereby only 50% developed a rather normal thylakoid membrane system. For comparison, mature foliage leaves had almost normal levels of chlorophyll but a severely reduced photosynthetic capacity. A proteome analysis of chloroplasts isolated from mature foliage leaves revealed that in the absence of WHIRLY1, the abundances of a considerable fraction of proteins were downregulated. The fraction included multiple nucleoid-associated proteins including components of the transcriptional apparatus. Furthermore, ribosomal proteins, subunits of pyruvate dehydrogenase, CLP protease, ATP synthase, Rubisco and chaperons/chaperonins were found to be downregulated. In conclusion, the characterization of the barley why1 mutant plants revealed that WHIRLY1 is not essential for chloroplast development. Rather, it ensures a fast and failure-free progression of chloroplast development by remodeling nucleoids, which serve as assembly platforms for a concerted workflow of the numerous processes required for chloroplast development. Gene expression analyses revealed that the disturbance of chloroplast development is signaled to the nucleus, indicating that WHIRLY1 is not part of the biogenic retrograde signaling of plastids.

plant biology↗

bioRxiv2024

Bacterial RNA promotes proteostasis through inter-tissuecommunication in C. elegans

Life expectancy has been increasing over the last decades, which is not matched by an increase in healthspan. Besides genetic composition, environmental and nutritional factors influence both health- and lifespan. Diet is thought to be a major factor for healthy ageing. Here, we show that dietary RNA species extend healthspan in C. elegans. Inherent bacterial-derived double stranded RNA reduces protein aggregation in a C. elegans muscle proteostasis model. This beneficial effect depends on low levels of systemic selective autophagy, the RNAi machinery in the germline, even when the RNA is delivered through ingestion in the intestine and the integrity of muscle cells. Our data suggest a requirement of inter-organ communication between the intestine, the germline and muscles. Our results demonstrate that bacterial-derived RNAs elicit a systemic response in C. elegans, which protects the animal from protein aggregation during ageing. We provide evidence that low stress levels are beneficial for healthspan. One-Sentence SummaryBacteria-derived dietary cues and inter-tissue communication promote proteostasis and fitness in C. elegans

cell biology↗

bioRxiv2024

Decoding the Baltic Sea's Past and Present: A simple Molecular Index for Ecosystem Assessment

Marginal sea ecosystems, such as the Baltic Sea, are severely affected by anthropogenic pressures, such as climate warming, pollution, and eutrophication, which increased in the course of the past century. Biodiversity monitoring data and assessment of environmental status in such systems have typically been carried out only for the past few decades, if at all, and knowledge on pre-impact stability and good ecological status is limited. An extension of monitoring time series can potentially be achieved through analyses of paleoecological records, e.g. for phytoplankton, which form the base of the food web and are highly susceptible to environmental changes. Within the phytoplankton community, dinoflagellates and diatoms play a significant role as primary producers, and their relative dominance in the spring bloom, calculated as Dia/Dino index, is used as an indicator for the environmental status of the Baltic Sea. To extend time series on the dominance patterns and include non-fossilized dinoflagellates, we here establish a simple droplet digital PCR (ddPCR) reaction on ancient DNA from sediment cores that decodes phytoplankton dynamics. We focus on two common spring bloom species, the diatom Skeletonema marinoi and the dinoflagellate Apocalathium malmogiense, for which we evaluate a DNA based dominance index. It performs very well in comparison to DNA metabarcoding and modern monitoring and can elucidate past species dominance across the past century in three basins of the Baltic and across millennia in two of these basins. For the past century, we see a dominance shift already starting before the mid-20th century in two of the Baltic Sea basins, thus substantially predating current monitoring programs. Shifts are only partly coeval among the cores and the index shows different degrees of stability. This pattern is confirmed across millennia, where a long-term stable relationship between the diatom and the dinoflagellate is observed in the Eastern Gotland Basin, while data from the Gulf of Finland bear testimony to a much more unstable relationship. This confirms that good ecological status based on the dominance pattern of diatoms and dinoflagellates must be established locally and exemplifies how sediment core DNA can be employed to extend monitoring data.

ecology↗

bioRxiv2024

The crystal structure of the Shetha Protein II (FeSII) from Azotobacter vinelandii suggests a domain swap

The Azotobacter vinelandii FeSII protein forms an oxygen resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin type ferredoxin, forming a dimer in solution. Previously, the crystal structure was solved (Schlesier et al., 2016), with 5 copies in the asymmetric unit. One copy is a normal adrenodoxin domain, forming a dimer with its crystallographic symmetry mate. The other four copies are in an "open" conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidised and reduced, and the large conformational change in the open configuration allowed binding to nitrogenase. We have independently solved the structure of FeSII in the same crystal form. Our positioning of the atoms in the unit cell is similar to the earlier report. However, our interpretation of the structure is different. We interpret the "open" conformation as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. We caution that crystal structures should be interpreted in terms of the contents of the entire crystal rather than one asymmetric unit.

biochemistry↗

bioRxiv2023

SGLT2 inhibitors attenuate endothelial to mesenchymal transition and cardiac fibroblast activation

Beneficial effects of sodium glucose co-transporter 2 inhibitors (SGLT2is) in cardiovascular diseases have been extensively reported leading to the inclusion of these drugs in the treatment guidelines for heart failure. However, molecular actions especially on non-myocyte cells remain uncertain. We observed dose-dependent inhibitory effects of two SGLT2is, dapagliflozin (DAPA) and empagliflozin (EMPA), on inflammatory signaling in human umbilical vein endothelial cells (HUVECs). Proteomics analyses and subsequent enrichment analyses discovered profound effects of these SGLT2is on proteins involved in mitochondrial respiration and actin cytoskeleton. Validation in functional oxygen consumption measurements as well as tube formation and migration assays revealed strong impacts of DAPA. Considering that most influenced parameters played central roles in endothelial to mesenchymal transition (EndMT), we performed in vitro EndMT assays and identified substantial reduction of mesenchymal and fibrosis marker expression as well as changes in cellular morphology upon treatment with SGLT2is. In line, human cardiac fibroblasts (HCFs) exposed to DAPA showed less proliferation, reduced ATP production, and decelerated migration capacity while less extensive impacts were observed upon EMPA. Mechanistically, sodium proton exchanger 1 (NHE1) as well as sodium-myoinositol cotransporter (SMIT) and sodium-multivitamin cotransporter (SMVT) could be identified as relevant targets of SGLT2is in non-myocyte cardiovascular cells as validated by individual siRNA-knockdown experiments. In summary, we found comprehensive beneficial effects of SGLT2is on human endothelial cells and cardiac fibroblasts. The results of this study therefore support a distinct effect of selected SGLT2i on non-myocyte cardiovascular cells and grant further insights into potential molecular mode of action of these drugs.

pharmacology and toxicology↗

bioRxiv2023

Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunisation in the pig lung

The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leucocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing haemagglutinin and nucleoprotein with or without IL-1{beta}. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1{beta} reduced the number of functionally active Treg. Second, influenza infection upregulated IFI6 in BAL cells, decreasing their susceptibility to virus replication in vitro. Our data provides a reference map of porcine BAL cells and reveals lasting immunological consequences of influenza infection and respiratory immunisation in a highly relevant large animal model for respiratory virus infection. Author SummaryPigs and humans have a similar anatomy and physiology. In humans, cells from lung-washes are used to study immune responses and it was shown that these cells are crucial in protection against respiratory diseases such as influenza and COVID-19. To better understand lung immunity, we compared genes expressed in cells of pig lung-wash to white blood cells, providing an atlas for future studies of immunity in the lung. We also tested a vaccine given to the lung containing IL-1{beta}, a strong immune activator that protects mice against influenza virus infection. However, although IL-1{beta} increased pig immune responses it did not protect pigs against infection. We also showed that the number of immune cells that dampen immune responses (regulatory T cells) is reduced. In addition, we demonstrated increased expression of a protein, IFI6, 21 days after infection showing that while immune cells in the lung have common properties, the invading organisms influence them significantly. Our study elucidates why some vaccines fail despite inducing powerful immune responses, emphasizes the need for caution when applying results from small animals like mice to humans, and indicates the importance of the pig as a model to study disease in humans and livestock.

immunology↗

bioRxiv2023

ALK signalling primes the DNA damage response sensitizing ALK-driven neuroblastoma to ATR inhibition

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and ALK, which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signalling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-day ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-day combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumour cells to a neuronal/Schwann cell lineage identity. Our results identify a unique ability of ATR inhibition to trigger neuroblastoma differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

cancer biology↗

bioRxiv2023

Finding the rhythm: Humans exploit nonlinear intrinsic dynamics of compliant systems in periodic interaction tasks

Everyday activities, like jumping on a trampoline or using a swing-stick, show that humans seemingly effortless support systems in their intrinsically preferred motions. Although this observation seems obvious, data-based evidence proving that humans indeed match system dynamics has been lacking, since everyday objects usually exhibit complex, nonlinear dynamics, which are in general not analytically solvable. Recent insights in the field of nonlinear mode theory and the development of a tool to compute modes for nonlinear systems enabled us to investigate human strategies to excite periodic motions in the interaction with nonlinear systems. In the setup of a high score game, participants interacted with differently configured virtual compliant double pendulum systems through a haptic joystick. Through the joystick, the user could command positions to a motor link connected to the pendulum by a spring and received resulting spring forces in return to convey the feeling of holding a flexible stick. The participants were asked to alternately hit two targets located on the computed nonlinear mode of the system as often as possible. All participants intuitively exploited the elasticity of the system by choosing a holding strategy of the motor link and only compensate for energy losses with small motions. In this way, the intrinsic dynamics of the double pendulum system were exploited leading to the predicted fast motions along the nonlinear modes. The human strategy stayed consistent when decreasing the target size or increasing the mass of the lower pendulum link, i.e., changing the dynamics. Consequently, the presented research provides data-based evidence that humans can indeed estimate the nonlinear dynamics of system and intuitively exploit these. Additionally, the introduction to nonlinear modes and ways to compute them could be a powerful tool for further investigations on human capabilities and strategies in periodic interactions with nonlinear systems. Author summaryWithout thinking about it, humans interact with a wide variety of objects in everyday life. This includes objects with very complex nonlinear dynamics such as flexible rods or ropes. Since it is not trivial to enforce trajectories far away from the systems intrinsic motions and frequencies, it is likely that humans explore and, whenever possible, exploit the natural dynamics of the system. By using a tool to predict the trajectories of systems with nonlinear dynamics, we collected human user data to validate this hypothesis for repetitive tasks with a virtual double pendulum. Indeed our research showed that humans supported mechanical systems in their respective intrinsic motions and were able to intuitively match the systems eigenfrequencies. In doing so, only little control effort and motion was needed from the users, which could aids to save energy and mental resources. Since both these aspects are limiting factors in continuous tasks, it seems to have an evolutionary benefit that humans are very capable in estimating and exploiting the natural dynamics of compliant systems and tune their own control strategy to be be synchronized to the controlled system.

neuroscience↗

bioRxiv2023

Heterologous sarbecovirus receptor binding domains as scaffolds for SARS-CoV-2 receptor binding motif presentation

Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center upon targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potently neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.

immunology↗

bioRxiv2023

The first European woolly rhinoceros mitogenomes retrieved from within cave hyena coprolites suggest long-lasting phylogeographic differentiation

The woolly rhinoceros (Coelodonta antiquitatis) is an iconic species of the Eurasian Pleistocene megafauna, which was abundant in Eurasia in the Pleistocene until its demise beginning approximately 10,000 years ago. Despite the early recovery of several specimens from well-known European archaeological sites, including its type specimen (Blumenbach 1799), no genomes of European populations were available so far, and all available genomic data originated exclusively from the Siberian population1. Using coprolites of cave hyenas (Crocuta crocuta spelea) recovered from Middle Palaeolithic layers of two caves in Germany (Bockstein-Loch and Hohlenstein-Stadel), we isolated and enriched predator and prey DNA to assemble the first European woolly rhinoceros mitogenomes, in addition to cave hyena mitogenomes. These mitogenomes of European woolly rhinoceros are genetically distinct from the Siberian woolly rhinoceros, and analyses of the more complete mitogenome suggests a split of the populations potentially coinciding with the earliest fossil records of wooly rhinoceros in Europe.

zoology↗

bioRxiv2023

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabAs binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

immunology↗

bioRxiv2023

A network-based model of Aspergillus fumigatus elucidates regulators of development and defensive natural products of an opportunistic pathogen

Aspergillus fumigatus is a notorious pathogenic fungus responsible for various harmful, sometimes lethal, diseases known as aspergilloses. Understanding the gene regulatory networks that specify the expression programs underlying this fungus diverse phenotypes can shed mechanistic insight into its growth, development, and determinants of pathogenicity. We used eighteen RNA-seq datasets (seventeen publicly available and one previously unpublished) of Aspergillus fumigatus to construct a comprehensive gene regulatory network resource. Our resource, named GRAsp (Gene Regulation of Aspergillus fumigatus), was able to recapitulate known regulatory pathways such as response to hypoxia, iron and zinc homeostasis, and secondary metabolite synthesis. Further, GRAsp was experimentally validated in two cases: one in which GRAsp accurately identified an uncharacterized transcription factor negatively regulating the production of the virulence factor gliotoxin and another where GRAsp revealed the bZip protein, AtfA, as required for fungal responses to microbial signals known as lipo-chitooligosaccharides. Our work showcases the strength of using network-based approaches to generate new hypotheses about regulatory relationships in Aspergillus fumigatus. We also unveil an online, user-friendly version of GRAsp available to the Aspergillus research community.

systems biology↗

bioRxiv2023

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding ofproteins with alpha helical domains

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we have quantified the expression of RPL39L in human pluripotent cells, cancer cell lines and tissue samples, and in mouse germ cells. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread proteasomal activity. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

molecular biology↗

bioRxiv2023

Genetic analysis of rab7 mutants in zebrafish

Vascular network formation requires the fusion of newly formed blood vessels and the emergence of a patent lumen between the newly established connections so that blood flow can start. Lumen formation has been shown to depend on the late endosomal/lysosomal pathway in various organs of animal tubular systems. Here, we identified a late endosomal/lysosomal vesicular fraction (Rab7/Lamp2) in early zebrafish angiogenic sprouts, which appears to contribute to apical membrane growth during lumen formation. To study the effect of the late endocytic pathway on vascular development, we generated mutant alleles for all three rab7 genes in zebrafish (rab7a, rab7ba, rab7bb). All rab7 genes are expressed in wild-type zebrafish and we did not detect any compensatory effects by the other rab7 isoforms in single knockout mutants, which were all viable. Only the triple mutant was lethal suggesting some functional redundancy. However, the different rab7 isoforms fulfil also at least partially independent functions because eggs laid from mothers lacking two rab7 (rab7a and/or rab7bb). showed reduced survival and contained enlarged yolk granules, suggesting maternal contribution of these two rab7. Finally, we observed minor effects on lumen formation in embryos which still express one copy of rab7. Our results support the notion that the late endocytic/lysosomal compartment contributes to lumen expansion.

genetics↗

bioRxiv2023

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins

During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.

cell biology↗

bioRxiv2023

Plastid fatty acid export (FAX) proteins in Arabidopsis thaliana - the role of FAX1 and FAX3 in growth and development

In plant cells, fatty acid (FA) synthesis occurs in the plastid stroma and thus requires subsequent FA export for lipid assembly in the endoplasmic reticulum. In this context, the membrane-intrinsic protein FAX1 has been described to mediate FA-export across the plastid inner envelope (IE). In Arabidopsis, FAX1 function is crucial for pollen cell wall formation, male fertility, cellular lipid homeostasis and plant biomass. Based on conserved structural features and sequence motifs, we here define the plant FAX-protein family localized in plastids. Besides their membrane-intrinsic domain, the plastid-targeted FAX1-FAX3 contain distinct N-terminal stretches. Among them, the apolipoprotein-like -helical bundle of FAX2 is the most prominent. Further, we could unequivocally localize FAX2 and FAX3 proteins together with FAX1 to the IE membrane of chloroplasts and develop a topology model for FAX1, FAX2, and FAX3. In yeast, all plastid FAX proteins - i.e. FAX1, FAX2, FAX3, FAX4 - can complement for FA-transport function. For FAX1 we show a new function in pollen tube growth as well as together with FAX3 in seed/embryo development and in rosette leaf growth. Since in comparison to fax1 single knockout mutants, fax1/fax3 double knockouts are seedling lethal and not able to develop mature rosette leaves, we conclude that FAX1 and FAX3 function together in vegetative leaf growth. HighlightWe define distinct structural features of plant FAX proteins in plastids and demonstrate that FAX1 and FAX3 have complementary functions in vegetative leaf growth.

plant biology↗