SEARCH · bioRxiv Science

Results for “cell biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,477 records · Page 82Linked to original sources

bioRxiv2024

Mechanical Profiling of Biopolymer Condensates through Acoustic Trapping

Characterizing the mechanical properties of single colloids is a central problem in soft matter physics. It also plays a key role in cell biology through biopolymer condensates, which function as membraneless compartments. Such systems can also malfunction, leading to the onset of a number of diseases, including many neurodegenerative diseases; the functional and pathological condensates are commonly differentiated by their mechanical signature. Probing the mechanical properties of biopolymer condensates at the single particle level has, however, remained challenging. In this study, we demonstrate that acoustic trapping can be used to profile the mechanical properties of single condensates in a contactless manner. We find that acoustic fields exert the acoustic radiation force on condensates, leading to their migration to a trapping point where acoustic potential energy is minimized. Furthermore, our results show that the Brownian motion fluctuation of condensates in an acoustic potential well is an accurate probe for their bulk modulus. We demonstrate that this framework can detect the change in the bulk modulus of polyadenylic acid condensates in response to changes in environmental conditions. Our results show that acoustic trapping opens up a novel path to profile the mechanical properties of soft colloids at the single particle level in a non-invasive manner with applications in biology, materials science, and beyond.

biophysics↗

bioRxiv2024

Cryo-EM structures of PP2A:B55-Eya3 and PP2A:B55-p107 define PP2A:B55 substrate recruitment

The phosphoprotein phosphatase (PPP) family of ser/thr phosphatases are responsible for the majority of all ser/thr dephosphorylation in cells. However, unlike their kinase counterpart, they do not achieve specificity via phosphosite recognition sequences, but instead bind substrates and regulators using PPP-specific short linear and/or helical motifs (SLiMs, SHelMs). Protein phosphatase 2A (PP2A) is a highly conserved PPP that regulates cell signaling and is a tumor suppressor. Here, we investigate the mechanisms of substrate and regulator recruitment to the PP2A:B55 holoenzyme to define how substrates and regulators engage B55 and understand, in turn, how these interactions direct phosphosite dephosphorylation. Our cryo-EM structures of PP2A:B55 bound to p107 (substrate) and Eya3 (regulator), coupled with biochemical, biophysical and cell biology assays, show that while B55 associates using a common set of interaction pockets, the mechanisms of substrate and regulator binding can differ substantially. This shows that B55-mediated substrate recruitment is distinct from that observed for PP2A:B56 and other PPPs. It also allowed us to identify the core B55 recruitment motif in Eya3 proteins, a sequence we show is conserved amongst the Eya family. Finally, using NMR-based dephosphorylation assays, we also showed how B55 recruitment directs PP2A:B55 fidelity, via the selective dephosphorylation of specific phosphosites. Because of the key regulatory functions of PP2A:B55 in mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites.

biochemistry↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗

bioRxiv2024

DNA polymerase Zeta is a robust reverse transcriptase

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol {eta} has reverse transcriptase activity, while others have suggested that the yeast TLS Pol {zeta} is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol {zeta} far surpasses Pol {eta} and all other DNA Pols in reverse transcriptase activity. We find that Pol {zeta} reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol {zeta} is the major DNA Pol that functions in the RNA templated DSB repair pathway.

biochemistry↗

bioRxiv2024

CRISPR/Cas9-based somatic knock-in of reporters in the avian embryo in ovo

Gene editing and protein tagging are at the heart of modern developmental and cell biology. The advent of CRISPR/Cas9 based methods offers the possibility to develop customized approaches for genomic manipulations in non-classical experimental models. Here, we show that highly efficient somatic knock-ins of long DNA fragments can be achieved in the developing chick neural tube in ovo. We compare different types of repair matrices and different methods for the delivery of the CRISPR/Cas9 machinery, and find that an all plasmid-based approach and short arms of homology provide an easy and efficient method to achieve high frequencies of knock-in insertions with virtually no background signal. We use this method to target fluorescent reporters and dynamically monitor the subcellular distribution of endogenously expressed tagged proteins, as well as to insert the Gal4-VP16 transcription factor or the Cre recombinase at specific loci to label neural sub-populations in the chick embryonic spinal cord. Finally, we show that the method can also be applied to target the epiblast and somitic mesoderm.

developmental biology↗

bioRxiv2024

DprA recruits ComM to facilitate recombination during natural transformation in Gram-negative bacteria

Natural transformation (NT) represents one of the major modes of horizontal gene transfer in bacterial species. During NT, cells can take up free DNA from the environment and integrate it into their genome by homologous recombination. While NT has been studied for >90 years, the molecular details underlying this recombination remain poorly understood. Recent work has demonstrated that ComM is an NT-specific hexameric helicase that promotes recombinational branch migration in Gram-negative bacteria. How ComM is loaded onto the post-synaptic recombination intermediate during NT, however, remains unclear. Another NT-specific recombination mediator protein that is ubiquitously conserved in both Gram-positive and Gram-negative bacteria is DprA. Here, we uncover that DprA homologs in Gram-negative species contain a C-terminal winged helix domain that is predicted to interact with ComM by AlphaFold. Using Helicobacter pylori and Vibrio cholerae as model systems, we demonstrate that ComM directly interacts with the DprA winged-helix domain, and that this interaction is critical for DprA to recruit ComM to the recombination site to promote branch migration during NT. These results advance our molecular understanding of recombination during this conserved mode of horizontal gene transfer. Furthermore, they demonstrate how structural modeling can help uncover unexpected interactions between well-studied proteins to provide deep mechanistic insight into the molecular coordination required for their activity. SIGNIFICANCE STATEMENTBacteria can acquire novel traits like antibiotic resistance and virulence through horizontal gene transfer by natural transformation. During this process, cells take up free DNA from the environment and integrate it into their genome by homologous recombination. Many of the molecular details underlying this process, however, remain incompletely understood. In this study, we identify a new protein-protein interaction between ComM and DprA, two factors that promote homologous recombination during natural transformation in Gram-negative species. Through a combination of bioinformatics, structural modeling, cell biological assays, and complementary genetic approaches, we demonstrate that this interaction is required for DprA to recruit ComM to the site of homologous recombination.

microbiology↗

bioRxiv2024

Virtual staining from bright-field microscopy for label-free quantitative analysis of plant cell structures

The applicability of a deep learning model for the virtual staining of plant cell structures using bright-field microscopy was investigated. The training dataset consisted of microscopy images of tobacco BY-2 cells with the plasma membrane stained with the fluorescent dye PlasMem Bright Green and the cell nucleus labeled with Histone-red fluorescent protein. The trained models successfully detected the expansion of cell nuclei upon aphidicolin treatment and a decrease in the cell aspect ratio upon propyzamide treatment, demonstrating its utility in cell morphometry. The model also accurately documented the shape of Arabidopsis pavement cells in both wild type and the bpp125 triple mutant, which has an altered pavement cell phenotype. Metrics such as cell area, circularity, and solidity obtained from virtual staining analyses were highly correlated with those obtained by manual measurements of cell features from microscopy images. Furthermore, the versatility of virtual staining was highlighted by its application to track chloroplast movement in Egeria densa. The method was also effective for classifying live and dead BY-2 cells using texture-based machine learning, suggesting that virtual staining can be applied beyond typical segmentation tasks. Although this method still has some limitations, its non-invasive nature and efficiency make it highly suitable for label-free, dynamic, and high-throughput analyses in quantitative plant cell biology.

plant biology↗

bioRxiv2024

Getting Started with Machine Learning for Experimental Biochemists and Other Molecular Scientists

Machine learning (ML) is rapidly gaining traction in many areas of experimental molecular science for elucidating relationships and patterns in large or complex data sets. Historically, ML was largely the preserve of those with specialized training in fields such as statistics or cheminformatics. Increasingly, however, ML methodologies are becoming part of the standard toolkit for experimental scientists across a range of disciplines. Lowering the barrier of entry to these ML techniques, for scientists without a significant background in computer science or statistics, is important to broadening access to these powerful methods. Here we provide detailed, step by step tutorials for performing four ML methods that are particularly useful for applications in biochemistry, cell biology, and drug discovery: hierarchical clustering, Principal Component Analysis (PCA), Partial Least-Squares Discriminant Analysis (PLSDA), and Partial Least-Squares Regression (PLSR). The protocols are written for the widely used software MATLAB, but no prior experience with MATLAB is required to use them. We include an explanation of each step, pitched at a level to be understood by investigators without any prior experience with ML, MATLAB, or any kind of coding. We also highlight the scientific issues pertaining to selecting and scaling the data to be analyzed, and describe controls to test the validity of the results obtained. Throughout, we emphasize the relationship between the scientific question and how to choose data and methods that will allow it to be addressed in a meaningful way. Our aim is to provide a basic introduction that will equip experimental chemical biologists and other chemical and biomedical scientists with the knowledge required to use ML to aid in the design of experiments, the formulation and data-driven testing of hypotheses, and the analysis of experimental data. Basic Protocol 1Clustering Basic Protocol 2Principal Component Analysis (PCA) Basic Protocol 3Partial Least Squares Discriminant Analysis (PLSDA) Basic Protocol 4Partial Least Squares Regression (PLSR)

biochemistry↗

bioRxiv2024

scCompass: An integrated cross-species scRNA-seq database for AI-ready

Emerging single-cell sequencing technology has generated large amounts of data, allowing analysis of cellular dynamics and gene regulation at the single-cell resolution. Advances in artificial intelligence enhance life sciences research by delivering critical insights and optimizing data analysis processes. However, inconsistent data processing quality and standards remain to be a major challenge. Here we propose scCompass, which provides a data quality solution to build a large-scale, cross-species and model-friendly single-cell data collection. By applying standardized data pre-processing, scCompass integrates and curates transcriptomic data from 13 species and nearly 105 million single cells. Using this extensive dataset, we are able to archieve stable expression genes (SEGs) and organ-specific expression genes (OSGs) in human and mouse. We provide different scalable datasets that can be easily adapted for AI model training and the pretrained checkpoints with state-of-the-art (SOTA) single-cell foundataion models. In summary, the AI-readiness of scCompass, which combined with user-friendly data sharing, visualization and online analysis, greatly simplifies data access and exploitation for researchers in single cell biology(http://www.bdbe.cn/kun).

bioinformatics↗

bioRxiv2024

Sequence specificity of an essential nuclear localization sequence in Mcm3

Proteins with nuclear localization sequences (NLSs) are directed into the cell nucleus through interactions between the NLS and importin proteins. NLSs are generally short motifs rich in basic amino acids; however, identifying NLSs can be challenging due to the lack of a universally conserved sequence. In this study, we characterized the sequence specificity of an essential and conserved NLS in Mcm3, a subunit of the replicative DNA helicase. Through mutagenesis and AlphaFold 3 (AF3) modeling, we demonstrate that the precise positioning of basic residues within the NLS is critical for nuclear transport of Mcm3 through optimal interactions with importin. Disrupting these interactions impairs the nuclear import of Mcm3, resulting in defective chromatin loading of MCM and poor cell growth. Our results provide a structure-guided framework for predicting and analyzing monopartite NLSs, which, despite lacking a single consensus sequence, retain key characteristics shared between the NLSs of Mcm3 and the SV40 large T antigen. Author SummaryTransporting proteins into and out of the cell nucleus is essential for chromosome-associated activities. Nuclear localization sequences (NLSs), short motifs rich in basic amino acids, are commonly found in nuclear proteins. NLSs work by interacting with importin, a key transport receptor responsible for recognizing and guiding NLS-containing proteins through the nuclear pore complex into the nucleus. Other than being rich in basic amino acids, NLSs generally lack a discernible consensus sequence, raising questions about how they specifically control nuclear transport through their interactions with importins. Through a detailed mutagenesis study of a conserved and essential NLS in Mcm3, a subunit of the replicative DNA helicase, we demonstrate that the use of AlphaFold 3 (AF3), alongside genetic, biochemical, and cell biological analyses, define key contacts between Mcm3s NLS and importin that are required for nuclear import of Mcm3.

microbiology↗

bioRxiv2024

Optical tomography reconstructing 3D motion and structure of multiple-scattering samples under rotational actuation

Optical Diffraction Tomography (ODT) has emerged as a powerful tool for imaging biological cells in a non-invasive and label-free manner. However, conventional approaches using ODT by varying the illumination are plagued by the missing cone problem, which introduces ambiguity and deteriorates the axial resolution in the reconstruction. Although utilizing object rotation has the potential to yield isotropic resolution, experimental control or prior retrieval of the rotational parameters is challenging. In this work, we demonstrate ODT of multiple-scattering samples undergoing variable rotational motion, unlocking the potential for isotropic resolution in non-contact systems. We introduce a comprehensive reconstruction method to jointly retrieve both sample and rotational motion in 3D. An interferometric setup enables the recording of amplitude and phase data while the object is rotated in a non-contact manner around one or more chosen axes in an acoustofluidic device. We evaluate the tomographic reconstruction performance of the method for clusters of micro-beads and highlight its suitability for biomedical application beyond single cells, demonstrating high-resolution reconstruction of dense cancer spheroids containing more than 100 cells.

bioengineering↗

bioRxiv2024

The complete genome sequence of the crayfish pathogen Candidatus Paracoxiella cheracis n.g. n.sp. provides insight into pathogenesis and the phylogeny of the Coxiellaceae family

The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii, represents the best characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis. Interestingly, we demonstrate that Candidatus P. cheracis encodes for a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologues of several important C. burnetii effectors including a homologue of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii. IMPORTANCEUsing long-read sequencing technology we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis, a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insight into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology manipulating effector proteins.

microbiology↗

bioRxiv2024

Facilitating Gene Editing in Human Lymphoma Cells Using Murine Ecotropic γ-Retroviruses

Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other pre-clinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic {gamma}-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat-1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic {gamma}-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research.

molecular biology↗

bioRxiv2024

Tuning mechanical milieux of tissue templates and their cellular inhabitants to guide mechanoadaptation

Mechanomics describes the adaptation of mesenchymal stem cells (MSCs) to their mechanical environment, via cytoskeletal remodeling, as well as changes in shape and volume, ultimately resulting in emergent lineage commitment. Here we elucidated effects of exogenous microtubule stabilization, using paclitaxel (PAX), on stem cells capacity to sense and adapt to changes in their local mechanical environment. We studied the interplay between the living, evolving cells and their mechanical environment using established experimental and computational tools for respective delivery and prediction of shape and volume changing stresses. Stiffened and volumetrically larger microtubule-stabilized MSCs and their experienced significantly different normal and shear stress compared to control cells when exposed to identical bulk laminar flow (0.2 dyn/cm2) for one hour. These spatiotemporal mechanical cues transduced to the nucleus via the cytoskeleton, triggering significantly different changes in gene expression indicative of emergent lineage commitment than those observed in control cells. Using a paired computational model, we further predicted a range of mechanoadaptation responses of microtubule-stabilized cells to scaled up flow magnitudes (1 and 2 dyn/cm2). Hence, MSCs adapt to as well as modulate their own mechanical environment via cytoskeletal remodeling and lineage commitment - microtubule stabilization changes not only MSCs mechanoadaptive machinery, their capacity to adapt, and their lineage commitment, but also their mechanical environment. Taken as a whole, these studies corroborate our working hypothesis that MSCs and their mechanoadaptive machinery serve as sensors and actuators, intrinsically linked to their lineage potential via mechanoadaptive feedback loops which are sensitive to exogenous modulation via biochemical and biophysical means. ClassificationBiological Systems Engineering, Computational Simulations, Cell Biology, Biophysics

bioengineering↗

bioRxiv2024

In situ structural analysis of mammalian cells using a 200 kV electron cryomicroscope: implications for research infrastructure.

BackgroundElectron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications. MethodsHere, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets. ResultsWe demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in [~]15 [A] resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications. DiscussionThis study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.

biophysics↗

bioRxiv2024

A Spatiotemporal Atlas of Mouse Gastrulation and Early Organogenesis to Explore Axial Patterning and Project In Vitro Models onto In Vivo Space

At the onset of murine gastrulation, pluripotent epiblast cells migrate through the primitive streak, generating mesodermal and endodermal precursors, while the ectoderm arises from the remaining epiblast. Together, these germ layers establish the body plan, defining major body axes and initiating organogenesis. Although comprehensive single cell transcriptional atlases of dissociated mouse embryos across embryonic stages have provided valuable insights during gastrulation, the spatial context for cell differentiation and tissue patterning remain underexplored. In this study, we employed spatial transcriptomics to measure gene expression in mouse embryos at E6.5 and E7.5 and integrated these datasets with previously published E8.5 spatial transcriptomics1 and a scRNA-seq2 atlas spanning E6.5 to E9.5. This approach resulted in a comprehensive spatiotemporal atlas, comprising over 150,000 cells with 88 refined cell type annotations as well as genome-wide transcriptional imputation during mouse gastrulation and early organogenesis. The atlas facilitates exploration of gene expression dynamics along anterior-posterior and dorsal-ventral axes at cell type, tissue, and organismal scales, revealing insights into mesodermal fate decisions within the primitive streak. Moreover, we developed a bioinformatics pipeline to project additional scRNA-seq datasets into a spatiotemporal framework and demonstrate its utility by analysing cardiovascular models of gastrulation3. To maximise impact, the atlas is publicly accessible via a user-friendly web portal empowering the wider developmental and stem cell biology communities to explore mechanisms of early mouse development in a spatiotemporal context.

developmental biology↗

bioRxiv2024

The inner integument controls embryo sac development and seed shape in Arabidopsis thaliana

The angiosperm ovule is characterized by the close association of the two generations, with the haploid female gametophyte or embryo sac being encapsulated by the diploid sporophyte, which usually forms two integuments. How the gametophyte and sporophyte coordinate their development has long been of interest. However, the function of the inner integument in embryo sac development has remained elusive. Here, we addressed this question. We applied a genetic ablation strategy to achieve an early block in inner integument outgrowth. We generated plants expressing BARNASE under the control of an early acting endothelium-specific promoter. Corresponding lines carried ovules lacking most of the inner integument. The genetic and cell biological data revealed that in the near absence of an inner integument embryo sac development is blocked at the mono-nuclear embryo sac stage in most pre-fertilization ovules. Approximately 10 percent of the ovules developed a functional embryo sac and underwent fertilization. Subsequent embryo and endosperm development appeared unperturbed and viable seeds were produced albeit of altered shape. Our results show that the inner integument plays an important role in early embryo sac development as well as ovule and seed shape, but is dispensable for embryo and endosperm development. Key words: embryo sac, embryo, endothelium, female gametophyte, integument, seed development HighlightGenetic ablation of the inner integument demonstrates its role in embryo sac development and its irrelevance for embryogenesis.

plant biology↗

bioRxiv2024

Advances in culturing of the sea star Patiria miniata

The use of the sea star Patiria miniata as a model system has produced groundbreaking advances in a disparate set of biomedical research fields, including embryology, immunology, regeneration, cell biology and evolution of development. Nonetheless, the life cycle of P. miniata has not yet been closed in the laboratory, precluding the generation of stable transgenic and mutant lines, which would greatly expand the toolset for experimentation with this model system. Rearing P. miniata in the laboratory has been challenging due to limited knowledge about metamorphosis cues, feeding habits of juveniles and their relatively long generation time. Here we report protocols to rear P. miniata embryos through sexual maturity in a laboratory setting. We provide detailed staging of early embryonic development at different temperatures, and show that larvae can be raised to competence in as little as 15 days. We find that retinoic acid induces metamorphosis effectively and present methods to rear juveniles on commercially available foods. We show that in a flow-through system, juveniles double in size every 2 months and reach sexual maturity in approximately 2 years. We report the first example of P. miniata raised through sexual maturity in a laboratory setting, paving the way for the generation of stable mutant sea star lines.

developmental biology↗