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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Simultaneous triple staining for detecting cell-type specific spatio-temporal distribution of cell wall materials in monocot roots

O_LIAnatomical and histochemical imaging of grass root systems relies on tissue sectioning and cell wall staining dyes because molecular reporter lines are limited for most organisms. C_LIO_LIDistinct staining dyes require variable incubation time and concentration across different tissues and organisms. As a result, staining with multiple dyes becomes time consuming or challenging. Here, we report a rapid method to perform simultaneous triple staining on a glass slide. The entire protocol requires [~]4 hours and a smaller volume of stain than traditional methods. C_LIO_LIWe tested this method using the roots of two economically important crops, Triticum aestivum (wheat) and Zea mays (maize), as proof of concept. We have also demonstrated the presence of exodermis in wheat roots. Additionally, we identified the formation of polar lignin caps in maize exodermis using our simultaneous triple staining method. C_LIO_LIThis method empowers a quantitative approach to cell biology by elucidating cell-type specific spatio-temporal distribution of cell wall materials in monocot root systems. C_LI

plant biology↗

bioRxiv2018

Three-dimensional nanostructure of an intact microglia cell

Microglia are non-neuronal cells of the myeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al. 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al. 2007; Paolicelli et al. 2011; Li et al. 2012; Aguzzi et al. 2013, Cunningham 2013, Cunningham et al. 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramon y Cajals group (Achucarro 1913, Rio-Hortega 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al. 2005; Nimmerjahn et al. 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al. 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012; Tremblay et al. 2012; Sipe et al. 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk 2006; Maco et al. 2013; Blazquez-Llorca et al. 2015, Bosch et al. 2015). Here we present the first volumetric ultrastructural reconstruction of an entire mouse hippocampal microglia using serial block face scanning electron microscopy (SBEM). Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.

neuroscience↗

bioRxiv2024

EyaHOST a modular genetic system for investigation of intercellular and tumor-host interactions in Drosophila melanogaster

Cell biology and genetic analysis of intracellular, intercellular and inter-organ interaction studies in animal models are key for understanding development, physiology, and disease. The MARCM technique can emulate tumor development by simultaneous clonal tumor suppressor loss-of-function generation coupled with GAL4-UAS-driven oncogene and marker expression, but the utility is limited for studying tumor-host interactions due to genetic constraints. To overcome this, we introduce EyaHOST, a novel system that replaces MARCM with the QF2-QUAS binary gene expression system under the eya promoter control, unleashing the fly community genome-wide GAL4-UAS driven tools to manipulate any host cells or tissue at scale. EyaHOST generates epithelial clones in the eye epithelium similar to MARCM. EyaHOST-driven RasV12 oncogene overexpression coupled with scribble tumor suppressor knockdown recapitulates key cancer features, including systemic catabolic switching and organ wasting. We demonstrate effective tissue-specific manipulation of host compartments such as neighbouring epithelial cells, immune cells, fat body, and muscle using fly avatars with tissue-specific GAL4 drivers. Organ-specific inhibition of autophagy or stimulation of growth-signaling through PTEN knockdown in fat body or muscle prevents cachexia-like wasting. Additionally, we show that RasV12, scribRNAi tumors induce caspase-driven apoptosis in the epithelial microenvironment. Inhibition of apoptosis by p35 expression in the microenvironment promotes tumor growth. EyaHOST offers a versatile modular platform for dissecting tumor-host interactions and other mechanisms involving intercellular and inter-organ communication in Drosophila. Highlights*eyes absent, eye disc-specific enhancer drives clonal KD recombinase flip-out activated QF2 expression in the larval eye epithelium for simultaneous QUAS-driven gain and loss-of-function analysis of gene function. *Clones are visualized by QUAS-tagBFP or QUAS-eGFP facilitating analysis of existing fluorescent reporters. *The GAL4-UAS system and existing genome-wide genetic tools are released to independently manipulate any cell population in the animal for cell biology, intercellular or inter-organ analysis for developmental, physiological, or disease model analysis. *Fly avatars for tumor-host interaction studies with multiple organs allow live monitoring and manipulation of tumors and organs in translucent larva.

cancer biology↗

bioRxiv2020

Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

biophysics↗

bioRxiv2025

LOBSTERS: a modular vector series with a diverse set of Cas nucleases and plasmid selection markers for multiplex genome editing in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants (RME1, TAO3, and MKT1) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae. By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance StatementO_LILOBSTERS integrates three PAM-diverse nucleases with seven plasmid selection markers, overcoming the single-marker limitation of previous yeast genome editing toolkits. C_LIO_LIThe system enables efficient simultaneous editing at multiple loci, demonstrated by functional tagging of essential proteins and recapitulation of quantitative trait variants. C_LIO_LIBy broadening the editable space and lowering barriers to complex genotype construction, LOBSTERS provides a widely applicable resource for yeast cell biology and synthetic biology. C_LI

molecular biology↗

bioRxiv2023

Chemotactic agents combining spatial and temporal gradient-sensing boost spatial comparison if they are large, slow, and less persistent

Biological cells and small organisms navigate in concentration fields of signaling molecules using two fundamental gradient-sensing strategies: spatial comparison of concentrations measured at different positions on their surface, or temporal comparison of concentrations measured at different locations visited along their motion path. It is believed that size and speed dictate which gradient-sensing strategy cells choose, yet this has never been formally proven. Using information theory, we investigate the optimal gradient-sensing mechanism for an ideal chemotactic agent that combines spatial and temporal comparison. We account for physical limits of chemo-sensation: molecule counting noise at physiological concentrations, and motility noise inevitable at the micro-scale. Our simulation data collapses onto an empirical power-law that predicts an optimal weighting of information as function of motility and sensing noise, demonstrating how spatial comparison becomes more beneficial for agents that are large, slow and less persistent. This refines and quantifies the previous heuristic notion. Our idealized model assuming unlimited information processing capabilities serves as a benchmark for the chemotaxis of biological cells.

biophysics↗

bioRxiv2024

Engineered Protein-G variants for plug-and-play applications

We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the "knobs into holes" strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent Bispecific T-cell Engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.

bioengineering↗

bioRxiv2025

Curated high-quality genomes of 39 diverse halophilic archaea

Archaea are widespread and ecologically important microorganisms, yet our understanding of their physiology and evolution is constrained by the limited number of complete genome assemblies available. Haloarchaea have emerged as model organisms for archaeal cell biology, virus-host interactions, and biotechnology. Despite their prominence in hypersaline environments and their potential for industrial applications, high-quality reference genomes remain scarce. Here, we present chromosome-level assemblies for 39 cultivable haloarchaeal strains for which no complete genomes were previously available. Using Oxford Nanopore sequencing, we obtained near-complete assemblies, with 38 strains resolving into single closed chromosomes and additional replicons such as plasmids captured largely in full. These genomes expand the available genomic resources for five haloarchaeal genera and provide a framework for comparative analyses of archaeal metabolism, genome organization, and mobile genetic elements. Given that many of these strains are natural hosts to diverse archaeal viruses, the genomes also represent a critical resource for advancing studies of virus-host interactions in archaea. Beyond fundamental insights into archaeal cell biology and evolution, this dataset will support the development of haloarchaeal model systems and facilitate the exploration of their biomolecules for biotechnological applications.

microbiology↗

bioRxiv2026

Intronised fluorophores improve reporter output and biosensor expression in plants

Genetically encoded biosensors are central to plant cell biology, but low reporter signal can limit their use. Here, we developed modular intronised fluorophores to improve reporter output in transient and stable plant expression systems. Matched nuclear-localised triple mCitrine and mScarlet3 reporters were compared with and without introduced introns in Nicotiana benthamiana and Arabidopsis thaliana. Intronisation increased fluorescence, with the magnitude of enhancement depending on the fluorophore and expression context. Immunoblot analyses supported increased reporter accumulation. In Arabidopsis, intronised reporters driven by the tissue-specific SCARECROW promoter, produced stronger fluorescence signals that remained detectable farther from the root tip than non-intronised versions. Intronised versions of the abscisic acid (ABA) biosensor ABACUS2 retained ABA responsiveness and showed a lower frequency of reduced fluorescence between generations. These modular components provide a practical toolbox for improving fluorescent reporter detection and maintaining usable biosensor expression, supporting their application in plant cell biology and quantitative imaging.

plant biology↗

bioRxiv2025

Creation and Validation of a Proteome-Wide Yeast Library for Protein Detection and Analysis

A significant challenge in cell biology is to uncover the function of uncharacterized proteins. Surprisingly a quarter of the proteome is still poorly understood even in the most well studied model organisms. Systematic methodologies, including the use of tagged protein collections, have emerged as a powerful approach to address this gap. Despite the availability of proteome- wide collections featuring various fused proteins, the impact of tag size on protein function highlighted the need for using minimally disruptive tags for functional genomic studies. To rise to this challenge, we have created a proteome-wide collection of yeast strains in which proteins are N-terminally tagged with the Hemagglutinin (HA) epitope. The library leverages the compact size of the HA tag to minimize drawbacks associated with larger tags while enabling efficient functional analysis. We showcase the potential uses of our library for systematically evaluating protein size, abundance and localization using an in vivo labeling approach. Our characterization underscores the potential utility of a proteome-wide HA-tagged library in revealing novel aspects of cell biology, providing an additional powerful tool for functional genomics.

molecular biology↗

bioRxiv2019

Selective Affimers Recognize BCL-2 Family Proteins Through Non-Canonical Structural Motifs

The BCL-2 family is a challenging set of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are desirable as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-xL, BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective and potent recognition of their target BCL-2 protein. For anti-apoptotic targets, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug bound like conformation. These results indicate Affimers can be used as alternative templates to inspire design of selective BCL-2 family modulators, and provide proof-of-concept for the elaboration of selective non-antibody binding reagents for use in cell-biology applications.

biochemistry↗

bioRxiv2022

Evolution of lineage-specific trafficking proteins and a novel post-Golgi trafficking pathway in Apicomplexa

The Organelle Paralogy Hypothesis (OPH) posits a mechanism to explain the evolution of non-endosymbiotically derived organelles, predicting that lineage-specific pathways organelles should result when identity-encoding membrane trafficking components duplicate and co-evolve. Here we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in the globally important lineage of parasites, the Apicomplexa. Using a new phylogenetic workflow, we are able to identify 18 novel paralogs of known membrane-trafficking machinery, the emergence of several of which correlate with the presence of new endomembrane organelles in apicomplexans or their larger lineage. Gene coregulation analysis of a large set of membrane-trafficking proteins in Toxoplasma both corroborate known molecular cell biological interactions between characterized machinery and suggest involvement of many of these new components into established pathways for biogenesis of or trafficking to the microneme and rhoptry invasion organelles. Moreover, focused molecular parasitological analysis of the apicomplexan Arf-like small GTPases, and the ArlX3 protein specifically, revealed a novel post-Golgi trafficking pathways involved in delivery of proteins to micronemes and rhoptries, with knock down demonstrating reduced invasion capacity. The totality of our data has identified an unforeseen post-Golgi trafficking pathway in apicomplexans and is consistent with the OPH mechanism acting to produce novel endomembrane pathways or organelles at various evolutionary stages across the Alveolate lineage. Significance statementThe mechanism of non-endosymbiotic organelle evolution has been relatively poorly explored and yet is relevant to many eukaryotic compartments, including the endomembrane system. The Organelle Paralogy Hypothesis predicts novel lineage-specific paralogs evolutionarily concurrent with emergence of new endomembrane organelles or pathways. By investigating this phenomenon in the apicomplexan parasites and their relatives, we identify and profile over a dozen new trafficking factors, several correlating with emergence of lineage-specific organelles. Cell biological study of one such factor demonstrates the existence of a novel post-Golgi trafficking pathway for components to the invasion organelles in the parasite Toxoplasma gondii. This work reveals how non-endosymbiotic organelle evolution has shaped cellular novelty in this lineage, relevant both to global health and fundamental evolutionary biology.

microbiology↗

bioRxiv2017

Transcriptomes and Raman spectra are linked linearly through a shared low-dimensional subspace

Raman spectroscopy is an imaging technique that can reflect whole-cell molecular compositions in vivo, and has been applied recently in cell biology to characterize different cell types and states. However, due to the complex molecular compositions and spectral overlaps, the interpretation of cellular Raman spectra have remained unclear. In this report, we compared cellular Raman spectra to transcriptomes of Schizosaccharomyces pombe and Escherichia coli, and provide firm evidence that they can be computationally connected and interpreted. Specifically, we find that the dimensions of high-dimensional Raman spectra and transcriptomes measured by RNA-seq can be effectively reduced and connected linearly through a shared low-dimensional subspace. Accordingly, we were able to reconstruct global gene expression profiles by applying the calculated transformation matrix to Raman spectra, and vice versa. Strikingly, highly expressed ncRNAs contributed to the Raman-transcriptome linear correspondence more significantly than mRNAs in S. pombe, which implies their major role in coordinating molecular compositions. This compatibility between whole-cell Raman spectra and transcriptomes marks an important and promising step towards establishing spectroscopic live-cell omics studies.

systems biology↗

bioRxiv2025

Complex interplay between gene deletions and the environment uncovers cellular roles for genes of unknown function in Escherichia coli

Phenotypic outcomes can be heavily affected by environmental factors. In this study, we exploited the previously observed nutrient-dependency of cell biological phenotypic features, captured by a cross-condition image-based profiling assay of Escherichia coli deletion strains, to examine this in more detail. We identified several general principles, including the existence of a spectrum of deviating phenotypes across nutrient conditions (i.e., from nutrient- or feature-specific to pleiotropic phenotypic deviations), limited conservation of phenotypic deviations across nutrient conditions (i.e., limited phenotypic robustness), and a subset of nutrient-independent phenotypic deviations (indicative of consistent genetic determinants of specific phenotypic features). In a subsequent step, we used this cross-condition dataset to identify five genes of unknown function of which the deletion displayed either nutrient-independent phenotypic deviations or phenotypic similarities to genes of known function: yibN, yaaY, yfaQ, ybiJ, and yijD. These genes showed different levels of phylogenetic conservation, ranging from conserved across the tree of life (yibN) to only present in some genera of the Enterobacterales (yaaY). Analysis of the structural properties of the proteins encoded by these y-genes, identification of structural similarities to other proteins, and the examination of their subcellular localization yielded new insights into their contribution to E. coli cell morphogenesis, cell cycle progression and cell growth. Together, our approach showcases how bacterial image-based profiling assays and datasets can serve as a gateway to reveal the function of uncharacterized proteins. ImportanceDespite unprecedented access to genomic information, predicting phenotypes based on genotypes remains notoriously difficult. One major confounding factor is the environment and its ability to modulate phenotypic outcomes. Another is the fact that a large fraction of protein-coding genes in bacterial genomes remains uncharacterized and have no known function. In this work, we use a large-scale cross-condition image-based profiling dataset to characterize nutrient-dependent phenotypic variability of E. coli deletion strains and exploit it to provide insight into the cellular role of genes of unknown function. Through our analysis, we identified five genes of unknown function that we subsequently further characterized by examining their phylogenetic conservation, predicted structural properties and similarities, and their intracellular localization. Combined, this approach highlights the potential of cross-condition image-based profiling, which extracts many cell biological phenotypic readouts across multiple conditions, to better understand nutrient-dependent phenotypic variability and uncover protein function.

microbiology↗

bioRxiv2022

Autophagy is associated with survival and resiliency of fibroblasts in long-term decaying post-mortem parenchymal tissue

Fibroblasts are spindle-shaped mesenchymal cells and an abundantly studied cell type that are easy to culture. Their adaptive response in culture conditions allows for use in many different cell biological experiments, including their utility in generating induced pluripotent stem cells. Despite extensive use of fibroblasts in cell and molecular biology and genetics experiments, fundamental evaluation of their resiliency and survival programs, in comparison with other cell types, is undetermined. Here, we demonstrate that fibroblasts exhibit remarkable survival capacity in post-mortem tissue decaying at room temperature and can be cultured from ear, tail tips, kidney, lung, fetal, and mammary tumor tissue after 12-hours of post-mortem tissue decay. Fibroblasts can be cultured from ear and lung tissue after 24-hours, and from ear after up to 120-hours of post-mortem tissue decay. Gene expression profiling of post-mortem lung tissue fibroblasts compared to fresh tissue cultured fibroblasts suggested a transition to a more quiescent phenotype with activation of nutrient scavenging pathways as evidenced by downregulation of genes associated with DNA replication, ribosomes, cell cycle, and spliceosomes as well as upregulation of genes associated metabolism, autophagy, and lysosomes. Measurement of light chain 3B (LC3B)-I/LC3B-II ratio and lysosomal-associated membrane protein (LAMP)-1 indicate that autophagy is increased in post-mortem fibroblasts, with evidence for potential increase in autolysosomes and senescence program. Our study provides evidence for the ability of normal fibroblasts to overcome extreme stress conditions and offers new insights into cell survival mechanisms and aging, with potential utility in tissue regeneration and repair.

cell biology↗

bioRxiv2021

DeepSea: An efficient deep learning model for automated cell segmentation and tracking

Dynamics and non-genetic heterogeneity are two fundamental characteristics of basic processes of life such as cell division or differentiation. Time-lapse microscopy is the only method that can directly capture the dynamics and heterogeneity of fundamental cellular processes at the singlecell level with high temporal resolution. Successful application of single-cell time-lapse microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. Recently, deep learning models have ushered in a new era in the quantitative analysis of microscopy images. However, integrated segmentation and tracking of single cells remain challenges for the analysis of time-lapse microscopy images. This work presents a versatile and trainable deep-learning software, termed DeepSea, that allows for both segmentation and tracking of single cells in sequences of phase-contrast live microscopy images. Our segmentation model can easily be trained to segment phase-contrast images of different cell types with higher precision than existing models. Our tracking model allows for quantification of dynamics of several cell biological features of individual cells, such as cell division cycle, mitosis, cell morphology, and cell size, with high precision using phase-contrast images. We showcase the application of DeepSea by analyzing cell size regulation in embryonic stem cells. Our findings show that embryonic stem cells exhibit cell size control in the G1 phase of the cell cycle despite their unusual fast division cycle. Our training dataset, user-friendly software, and code are available here https://deepseas.org.

systems biology↗

bioRxiv2018

A method for genome editing in the anaerobic magnetotactic bacterium Desulfovibrio magneticus RS-1

ABSTRACTMagnetosomes are complex bacterial organelles that serve as model systems for studying cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacterial Magnetospirillum spp. that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have also been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing Deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified ten genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed many interesting mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration has not been feasible due to low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.\n\nIMPORTANCEMagnetotactic bacteria (MTB) are a group of organisms that form small, intracellular magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membrane, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization as well as potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply-branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from the cubooctahedral-shaped magnetite crystals of the genetically tractable Alphaproteobacteria MTB. Here, we present a method for genome editing in the anaerobic Deltaproteobacterium Desulfovibrio magneticus RS-1, the first cultured representative of the deeply-branching MTB. This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

microbiology↗

bioRxiv2021

High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress

Protein kinases regulate fundamental aspects of cell biology in all eukaryotes, making them attractive chemotherapeutic targets in Apicomplexan parasites such as the causative agents of malaria (Plasmodium spp.) and toxoplasmosis (Toxoplasma gondii). However, the precise roles of individual parasite kinases cannot be inferred simply from sequence identity, due to rewiring of signaling pathways and the shifting repertoire of kinases across species. To systematically examine the parasite kinome, we developed a high-throughput (HiT) CRISPR-mediated tagging strategy to endogenously label all predicted cytosolic protein kinases with a synthetic sequence encoding the minimal auxin-inducible degron (mAID) linked to a fluorophore and epitope tag. The system enables the assembly of thousands of tagging vectors from synthetic sequences in a single reaction and the pooled generation of mutants to examine kinase localization and function. We examined the phenotypes associated with kinase knock-down in 1,160 arrayed clones by replica-plating in the presence or absence of auxin and found broad defects across the lytic cycle for 109 clonal isolates, assigning localizations to 39 proteins, and associating 15 kinases within 6 distinct morphological phenotypes. The relative fitness of tagged alleles was also examined by tracking the relative abundance of individual guide RNAs as parasite populations progressed through the lytic cycle, in the presence or absence of auxin. Pooled screening had a high predictive value and differentiated between delayed and acute death. Demonstrating the value of this resource, we identified a novel kinase associated with delayed death as a novel regulator of invasion and egress. We call the previously unstudied kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores at key moments during the lytic cycle. Despite having a similar kinase domain to the mammalian PDK1, SPARK lacks the canonical lipid-binding domain and we find no indication SPARK positively regulates other AGC kinases, suggesting a rewiring of signaling pathways to accommodate parasite adaptations. The HiT vector screening system extends the applications of genome-wide screens into complex cellular phenotypes, providing a scalable and versatile platform for the dissection of apicomplexan cell biology.

microbiology↗