Biology subjects

Sato, Y.

Publications and source records attributed to Sato, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Detecting frequency-dependent selection through the effects of genotype similarity on fitness components

Frequency-dependent selection (FDS) drives an evolutionary regime that maintains or disrupts polymorphisms. Despite the increasing feasibility of genetic association studies on fitness components, there are a few methods to uncover the loci underlying FDS. Based on a simplified model of pairwise genotype-genotype interactions, we propose a linear regression that can infer FDS from observed fitness. The key idea behind our method is the inclusion of genotype similarity as a pseudo-trait in selection gradient analysis. Single-locus analysis of Arabidopsis and damselfly data could detect known negative FDS on visible polymorphism that followed Mendelian inheritance with complete dominance. By extending the singlelocus analysis to genome-wide association study (GWAS), our simulations showed that the regression coefficient of genotype similarity can distinguish negative or positive FDS without confounding other forms of balancing selection. Field GWAS of the branch number further revealed that negative FDS, rather than positive FDS, was enriched among the top-scoring single nucleotide polymorphisms (SNPs) in Arabidopsis thaliana. These results showed the wide applicability of our method for FDS on both visible polymorphism and genome-wide SNPs. Our study provides an effective method for selection gradient analysis to understand the maintenance or loss of polymorphism.

evolutionary biology↗

bioRxiv2022

Hypoxia causes pancreatic β-cell dysfunction by activating a transcriptional repressor BHLHE40

Hypoxia can occur in pancreatic {beta}-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on {beta}-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human {beta}-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or in the {beta}-cells of ob/ob mice reversed the insulin secretion. Mechanistically, BHLHE40 represses expression of Mafa, which encodes the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic {beta}-cells was recovered by MAFA expression. Collectively, this work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in {beta}-cells and its implication in the {beta}-cell dysfunction in type 2 diabetes.

biochemistry↗

bioRxiv2022

Nanog organizes transcription bodies

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis, to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription, and independently of RNA accumulation. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies. HIGHLIGHTSO_LITranscription factors cluster prior to, and independently of transcription C_LIO_LINanog organizes transcription bodies: it is required for the clustering of Sox19b as well as RNA polymerase II C_LIO_LIThis organizing activity requires its DNA binding domain as well as one of its intrinsically disordered regions C_LIO_LITranscription elongation results in the disassembly of transcription factor clusters C_LI

cell biology↗

bioRxiv2022

Intranasal vaccination induced cross-protective secretory IgA antibodies against SARS-CoV-2 variants with reducing the potential risk of lung eosinophilic immunopathology

To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-{gamma}-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.

microbiology↗

bioRxiv2022

"Two-story building" of a ctenophore comb plate provides structural and functional integrity for motility of giant multicilia

Comb plates (CPs) are large compound cilia uniquely seen in a basal metazoan group of ctenophores.1-3 Tens of thousands of cilia are bundled in a CP via structures connecting adjacent cilia, called compartmenting lamella (CL), which are the basis for the structural iridescent color and the coordination of ciliary movement of the CP.4-6 We previously reported the first component of CL, CTENO64, and found that it was convergently acquired in ctenophores and was essential for determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of CL over the entire length has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of the CL, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene with morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region, but did not affect either the formation of CP or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of CP along a comb row was lost in the CTENO189 morphants. The morphant CP showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms, becoming a symmetric flagellar-type. Our findings demonstrate a "two-story building" of CP, comprising the proximal CL, as the building foundation that rigidly fixes the ciliary orientation. The distal CL would reinforce the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.

cell biology↗

bioRxiv2022

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

molecular biology↗

bioRxiv2021

The UV RESISTANCE LOCUS 8-Mediated UV-B Response Is Required Alongside CRYPTOCHROME1 For Plant Survival Under Sunlight In The Field

As sessile organisms, plants are subjected to fluctuating sunlight including potentially detrimental ultraviolet-B radiation (UV-B). In Arabidopsis thaliana, experiments under controlled conditions have shown that UV RESISTANCE LOCUS 8 (UVR8) controls photomorphogenic responses for acclimation and tolerance to UV-B; however, its long-term impacts on plant performance remain poorly understood in naturally fluctuating environments. Here we quantified the survival and reproduction of different Arabidopsis mutant genotypes in diverse field and laboratory conditions. We found that uvr8 mutants produced more fruits than wild type in growth chambers with artificial low UV-B conditions but not in natural field conditions. Importantly, independent double mutants of UVR8 and the blue-light photoreceptor gene CRYPTOCHROME 1 (CRY1) in two genetic backgrounds showed a drastic reduction in fitness in the field. UV-B attenuation experiments in field conditions and supplemental UV-B in growth chambers demonstrated that UV-B caused the conditional cry1 uvr8 lethality phenotype. RNA sequencing in different conditions revealed a large number of genes with statistical interaction of UVR8 and CRY1 mutations in the presence of UV-B in the field. Among them, Gene Ontology analysis identified enrichment of categories related to UV-B response, oxidative stress, photoprotection and DNA damage repair. Our study demonstrates the functional importance of the UVR8-mediated response across life stages in natura, which is partially redundant with CRY1, and provides an integral picture of gene expression associated with plant environmental responses under diverse environmental conditions.

plant biology↗

bioRxiv2021

Differential regulation of degradation and immune pathways underlies adaptation of the symbiotic nematode Laxus oneistus to oxic-anoxic interfaces

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also upregulated when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worms Toll- like innate immunity pathway and several immune effectors (e.g., Bacterial Permeability Increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires oxidative phosphorylation and by reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

genomics↗

bioRxiv2021

Bacterial growth in multicellular aggregates leads to the emergence of complex lifecycles

In response to environmental stresses such as starvation, many bacteria facultatively aggregate into multicellular structures that can attain new metabolic functions and behaviors. Despite the ubiquity and relevance of this form of collective behavior, we lack an understanding of how the spatiotemporal dynamics of aggregate development emerge from cellular physiology. Here, we show that the coupling between growth and spatial gradient formation leads to the emergence of a complex lifecycle, akin to those known for multicellular bacteria. Under otherwise carbon-limited growth conditions, the marine bacterium Vibrio splendidus 12B01 forms multicellular groups to collectively harvest carbon from the brown-algal polysaccharide alginate. This is achieved during growth on dissolved alginate polymer through formation of spherical, clonal clusters of cells that grow up to 40 {micro}m in radius. Clusters develop striking spatial patterning as they grow due to phenotypic differentiation of sub-populations into a shell of static cells surrounding a motile core. Combining in situ measurements of cell physiology with transcriptomics, we show that shell cells express adhesive type IV pili, while motile core cells express carbon storage granules. The emergence of shell and core phenotypes is cued by opposing gradients of carbon and nitrogen that form within cell clusters due to local metabolic activity. Eventually, the shell ruptures, releasing the carbon-storing core, and we show that carbon-storing cells more readily propagate on alginate than non-carbon storing cells. We propose that phenotypic differentiation promotes the resilience of 12B01 groups by enabling clonal groups to grow larger and propagate more effectively. Phenotypic differentiation may be a widespread, but overlooked, strategy among bacteria to enhance resilience in the context of resource limitation.

microbiology↗

bioRxiv2021

Single-cell profiling of transcriptome and histone modifications with EpiDamID

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, including various combinations of transcriptome, genome and epigenome. However, comprehensive profiling of the histone post-translational modifications that influence gene expression at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of genetically engineered proteins. By fusing Dam to single-chain variable fragment antibodies, engineered chromatin reader domains, or endogenous chromatin-binding proteins, we render the DamID technology and all its implementations compatible with the genome-wide identification of histone post-translational modifications. Importantly, this enables the joint analysis of chromatin marks and transcriptome in a variety of biological systems at the single-cell level. In this study, we use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. We further demonstrate the applicability of this method to in vivo systems by mapping H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specifically in the notochord. Overall, EpiDamID is a new addition to a vast existing toolbox for obtaining systematic insights into the role of chromatin states during dynamic cellular processes.

genomics↗

bioRxiv2021

Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness

AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling. SignificanceCanine models of inherited retinal diseases have informed and advanced AAV gene therapies targeting specific cells, including photoreceptors in the outer retina, for treatment of blinding diseases in human patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the mid-retina remains under-developed. Using a new LRIT3 mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of a cell-specific AAV capsid and promotor to specifically target ON-BCs for gene delivery. Notably, subretinal injection of AAV-LRIT3 vector demonstrated safety and efficacy with robust and stable rescue of ERG signals and night vision up to 1 year, paving the way for clinical trials in CNSB patients.

neuroscience↗

bioRxiv2021

Proliferation maintains the undifferentiated status of stem cells: the role of the planarian cell cycle regulator Cdh1

The coincidence of cell cycle arrest and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since it possesses a quite simple cell cycle regulation in which cdh1 is the only factor that arrests the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the ablation of cdh1 caused pronounced propagation of the stem cells, the progenitor and differentiated cells were decreased. Further analysis indicated that the stem cells without cdh1 expression did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle arrest. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation. Summary statementBy using planarians, which have quite simple cell cycle regulation, we revealed that stem cells with cell cycle progression could not undergo differentiation even though an induction signal was activated.

developmental biology↗

bioRxiv2021

Using a two-stage convolutional neural network to rapidly identify tiny herbivorous beetles in the field

Recently, deep convolutional neural networks (CNN) have been adopted to help beginners identify insect species from field images. However, the application of these methods on the identification of tiny congeneric species moving across heterogeneous background remains difficult. To enable rapid and automatic identification in the field, we customized a method involving real-time object detection of two Phyllotreta beetles. We first performed data augmentation using transformations, syntheses, and random erasing of the original images. We then proposed a two-stage method for the detection and identification of small insects based on CNN, where YOLOv4 and EfficientNet were used as a region proposal network and a re-identification method, respectively. Evaluation of the model revealed that one-step object detection by YOLOv4 alone was not precise (Precision = 0.55) when classifying two species of flea beetles and background objects. In contrast, the two-step CNNs improved the precision (Precision = 0.89) with moderate accuracy (F-measure = 0.55) and acceptable speed (ca. 5 frames per second for full HD images) of detection and identification of insect species in the field. Although real-time identification of tiny insects remains a challenge in the field, our method aids in improving small object detection on a heterogeneous background.

ecology↗

bioRxiv2021

Oxidized alkyl phospholipids stimulate sodium transport in proximal tubules via PPAR-dependent pathway

The pleiotropic effects of oxidized phospholipids (oxPLs) have been identified. 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC), an oxPL formed from alkyl phosphatidylcholines, is a potent peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist. Although it has been reported that thiazolidinediones can induce volume expansion by enhancing renal sodium and water retention, the role of azPC, an endogenous PPAR{gamma} agonist, in renal transport functions is unknown. In the present study, we investigated the effect of azPC on renal proximal tubule (PT) transport using isolated PTs and kidney cortex tissues. We showed that azPC rapidly stimulated Na+/HCO3- cotransporter 1 activity and luminal Na+/H+ exchanger (NHE) activities in a dose-dependent manner, at submicromolar concentrations, in isolated PTs from rats and humans. Additionally, the stimulatory effects were completely blocked by a specific PPAR{gamma} antagonist, 2-chloro-5-nitro-N-phenylbenzamide (GW9662), and a mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor, PD98059. Treatment with an siRNA against PPAR{gamma} significantly suppressed the expression of PPAR{gamma} mRNA, and it completely blocked the stimulation of both Na+/HCO3- cotransporter 1 and NHE activities by azPC. Moreover, azPC induced extracellular signal-regulated kinase (ERK) phosphorylation in rat and human kidney cortex tissues, and the induced ERK phosphorylation by azPC was completely suppressed by GW9662 and PD98059. These results suggest that azPC stimulates renal PT sodium-coupled bicarbonate transport via the PPAR{gamma}/MEK/ERK pathway. The stimulatory effects of azPC on PT transport may be partially involved in the development of volume expansion.

physiology↗

bioRxiv2021

q-mer analysis: a generalized method for analyzing RNA-Seq data.

RNA-Seq data are usually summarized by counting the number of transcript reads aligned to each gene. However, count-based methods do not take alignment information, where and how each read was mapped in the gene, into account. This information is essential to characterize samples accurately. In this study, we developed a method to summarize RNA-Seq data without losing alignment information. To include alignment information, we introduce "q-mer analysis," which summarizes RNA-Seq data with 4q kinds of q-length oligomers. Using publicly available RNA-Seq datasets, we demonstrate that at least q [greater double equals] 9 is required for capturing alignment information in Homo sapiens. Furthermore, principal component analysis showed that q-mer analysis with q = 14 linearly distinguished samples from controls, while a count-based method failed. These results indicate that alignment information is essential to characterize transcriptomics samples. In conclusion, we introduce q-mer analysis to include alignment information in RNA-Seq analysis and demonstrate the superiority of q-mer analysis over count-based methods in that q-mer analysis can distinguish case samples from controls. Combining RNA-Seq research with q-mer analysis could be useful for identifying distinguishing transcriptomic features that could provide hypotheses for disease mechanisms.

bioinformatics↗

bioRxiv2021

Highly variable fidelity drives symbiont community composition in an obligate symbiosis

Multipartite symbioses, which involve intimate interactions among three or more species, have evolved independently numerous times in convergent evolution, and enable functional complexity and ecological flexibility. How stability is maintained in such relationships remains, however, poorly understood, and can only be fully understood by integrating processes across both micro- and macroevolutionary scales. In this study, we bridge these scales by examining the symbiotic communities of gutless marine oligochaetes - annelid worms that have become obligately dependent on their bacterial partners for nutrition and waste removal over the past 150 million years. Using metagenomic sequencing of 231 individuals from 63 host species, collected across 17 marine environments worldwide, we reconstructed host and symbiont phylogenies to investigate the composition, specificity, and evolutionary history of their symbiotic communities from the strain to genus level. Our results show that each gutless oligochaete harbors up to 10 symbiont species drawn from a restricted pool of 33 marine bacterial genera. Community composition was strongly shaped by host species at 90% explanatory power, and much less by geography or environment. Symbiont communities were highly stable within host species but shifted rapidly at short macroevolutionary scales as host species diverged. Ancestral state reconstructions revealed that the primary symbiont, Candidatus Thiosymbion, was an early and persistent acquisition, while secondary symbionts were gained multiple times through convergent evolution, enhancing the diversity and metabolic flexibility of the symbiotic communities. By linking fine-scale microevolutionary dynamics with broader macroevolutionary patterns, our study reveals how gutless oligochaetes achieve both stability and flexibility in their symbiotic partnerships - enabling long-term host dependency while driving innovation and adaptation. This evolutionary trajectory may underpin the ecological success and diversification of gutless oligochaetes in marine sediments, and may help explain the repeated emergence and persistence of other multipartite associations.

evolutionary biology↗

bioRxiv2021

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription "factories" in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters. SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

cell biology↗

bioRxiv2021

Translation of cellular protein localization by generative adversarial network

The protein localization in cells had been analyzed by the fluorescent labeling by indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localizations between different proteins had not been analyzed by artificial intelligence. In this study, we applied the generative adversarial network (GAN) to generate the protein localizations each other, in which the generation was dependent on the types of cells and the relationships between the proteins. Lamellipodia are one of the actin-dependent subcellular structures involved in cell migration and are mainly generated by the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 2 (WAVE2) and the membrane remodeling I-BAR domain protein IRSp53. Focal adhesions are another actin-based structure that contains vinculin protein and are essential for cell migration. In contrast, microtubules are not thought to be directly related to actin filaments. The GAN was trained using images of actin filaments paired with WAVE2, vinculin, IRSp53, and microtubules. Then, the generated images of WAVE2, vinculin, and IRSp53 by the GAN showed high similarity to the real images of WAVE2, vinculin, and IRSp53, respectively. However, the microtubule images generated from actin filament images were inferior, corroborating that the microscopic images of actin filaments provide more information about actin-related protein localization. Collectively, this study suggests that the image translation by the GAN can predict the localization of functionally related proteins.

cell biology↗