Biology subjects

Aoki, K.

Publications and source records attributed to Aoki, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade

Anhydrobiosis is one of the most extensively studied forms of cryptobiosis that is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades response to desiccation. However, the molecular mechanisms underlying these protein functions remain to be fully elucidated. In this study, we perfomed in vitro and in vivo characterizations of the self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. Our in vitro observations showed that CAHS1 proteins homo-oligomerized via the C-terminal -helical region and formed a hydrogel as their concentration increased, and that these molecular assembling processes were reversible. Furthermore, our in vivo observations demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into the protective mechanisms involved in the anhydrobiosis of tardigrades.

biochemistry↗

bioRxiv2021

Redundant and specific roles of EGFR ligands in the ERK activation waves during collective cell migration of MDCK cells

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves by gene knockout. Four of the seven known EGFR ligands are expressed in MDCK cells. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGF) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Therefore, we knocked out the EGFR ligand genes in decreasing order of expression. ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked-out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout Nrg1, a ligand for ErbB3 and ErbB4, and found that Nrg1 deficiency induced growth arrest in the absence of all four EGFR ligand genes expressed in MDCK cells. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

cell biology↗

bioRxiv2021

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from bacteriophytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that of iRFP attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

cell biology↗

bioRxiv2021

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.

cell biology↗

bioRxiv2021

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

synthetic biology↗

bioRxiv2021

RaptGen: A variational autoencoder with profile hidden Markov model for generative aptamer discovery

Nucleic acid aptamers are generated by an in vitro molecular evolution method known as systematic evolution of ligands by exponential enrichment (SELEX). A variety of candidates is limited by actual sequencing data from an experiment. Here, we developed RaptGen, which is a variational autoencoder for in silico aptamer generation. RaptGen exploits a profile hidden Markov model decoder to represent motif sequences effectively. We showed that RaptGen embedded simulation sequence data into low-dimension latent space dependent on motif information. We also performed sequence embedding using two independent SELEX datasets. RaptGen successfully generated aptamers from the latent space even though they were not included in high-throughput sequencing. RaptGen could also generate a truncated aptamer with a short learning model. We demonstrated that RaptGen could be applied to activity-guided aptamer generation according to Bayesian optimization. We concluded that a generative method by RaptGen and latent representation are useful for aptamer discovery. Codes are available at https://github.com/hmdlab/raptgen.

bioinformatics↗

bioRxiv2020

Experimental evaluation of thermodynamic cost and speed limit in living cells via information geometry

Chemical reactions are responsible for information processing in living cells, and thermodynamic trade-off relations can explain their accuracy and speed. Its experimental test in living cells had not existed despite its importance because it is hard to justify sample size sufficiency. This paper reports the first experimental test of the thermodynamic trade-off relation, namely the thermodynamic speed limit, in living systems at the single-cell level where the sample size is relatively small. Due to the information-geometric approach, we can demonstrate the thermodynamic speed limit for the extracellular signal-regulated kinase phosphorylation using time-series fluorescence imaging data. Our approach quantifies the intrinsic speed of cell proliferation and can potentially apply other signal transduction pathways to detect their information processing speed. One-Sentence SummaryExperimental measurement of information thermodynamic speed by fluorescence imaging in living cells

biophysics↗

bioRxiv2020

A self-exciting point process to study multi-cellular spatial signaling patterns

Multi-cellular organisms rely on spatial signaling among cells to drive their organization, development, and response to stimuli. Several models have been proposed to capture the behavior of spatial signaling in multi-cellular systems, but existing approaches fail to capture both the autonomous behavior of single cells and the interactions of a cell with its neighbors simultaneously. We propose a spatiotemporal model of dynamic cell signaling based on Hawkes processes--self-exciting point processes--that model the signaling processes within a cell and spatial couplings between cells. With this cellular point process (CPP) model, we capture both the single-cell protein bursting rate and the magnitude and duration of signaling between cells relative to spatial locations. Furthermore, our model captures tissues composed of heterogeneous cell types with different bursting rates and signaling behaviors across multiple signaling proteins. We apply our model to epithelial cell systems that exhibit a range of autonomous and spatial signaling behaviors basally and under pharmacological exposure. Our model identifies known drug-induced signaling deficits, characterizes differences in signaling across a wound front, and generalizes to multi-channel observations.

cell biology↗

bioRxiv2020

Virus-Receptor Interactions of Glycosylated SARS-CoV-2 Spike and Human ACE2 Receptor

The current COVID-19 pandemic is caused by the SARS-CoV-2 betacoronavirus, which utilizes its highly glycosylated trimeric Spike protein to bind to the cell surface receptor ACE2 glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatic analyses of natural variants and with existing 3D-structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation that, taken together, can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

biochemistry↗

bioRxiv2020

Development of optogenetic tools to manipulate cell cycle checkpoints

In order to understand the systematic regulation of the cell cycle, we need more precise tools for cell-cycle perturbation. Optogenetics is a powerful technique for precisely controlling cellular signaling at higher spatial and temporal resolution. Here, we report optogenetic tools for the rapid and reversible control of cell-cycle checkpoints with a red/far-red light photoreceptor, phytochrome B (PhyB). We established fission yeast cells producing phycocyanobilin as a chromophore of PhyB, and demonstrated light-dependent protein recruitment to the plasma membrane, nucleus, and kinetochore. Using this system, we developed optogenetic manipulation of the cell cycle in two ways: the Opto-G2/M checkpoint triggered G2/M cell cycle arrest in response to red light, and Opto-SAC induced a spindle assembly checkpoint (SAC) in response to red light and then quickly released the SAC by far-red light.

cell biology↗

bioRxiv2020

Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

Oncogene addiction is a cellular property by which cancer cells become highly dependent on the expression of oncogenes for their survival. Oncogene addiction can be exploited to design molecularly targeted drugs that kill only cancer cells by inhibiting the specific oncogenes. Genes and cell lines exhibiting oncogene addiction, as well as the mechanisms by which cell death is induced when addicted oncogenes are suppressed, have been extensively studied. However, it is still not fully understood how oncogene addiction is acquired in cancer cells. Here, we take a synthetic biology approach to investigate whether oncogenic mutation or oncogene expression suffices to confer the property of oncogene addiction to cancer cells. We employed human mammary epithelium-derived MCF-10A cells expressing the oncogenic KRAS or BRAF. MCF-10A cells harboring an oncogenic mutation in a single-allele of KRAS or BRAF showed weak tumorigenic activity, but no characteristics of oncogene addiction. MCF-10A cells overexpressing oncogenic KRAS demonstrated the tumorigenic activity, but MCF-10A cells overexpressing oncogenic BRAF did not. Neither cell line exhibited any oncogene addiction properties. These results indicate that the introduction of oncogenic mutation or the overexpression of oncogenes is not sufficient for cancer cells to acquire oncogene addiction, and that oncogene addiction is not associated with tumorigenic potential.

cancer biology↗

bioRxiv2020

A genetically encoded red fluorescence dopamine biosensor enables dual imaging of dopamine and norepinephrine

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in finely controlling output functions is currently challenging, primarily due to a lack of techniques to visualize spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed R-GenGAR-DA. More specifically, a circular permutated red fluorescent protein (cpmApple) was inserted into the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated reasonable dynamic range ({Delta}F/F0 = -50%) and DA affinity (EC50 = 0.7 {micro}M) as well as the highest selectivity for DA over NE (143-fold) amongst available DA biosensors. Due to its high selectivity, R-GenGAR-DA1.2 allowed dual-color fluorescence live imaging for monitoring DA and NE, combined with the existing green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (>350-fold) in HeLa cells and hippocampal neurons grown from primary culture. By enabling precise measurement of DA, as well as simultaneous visualization of DA and NE, the red-DA biosensor R-GenGAR-DA1.2 is promising in advancing our understanding of the interplay between DA and NE in organizing key brain functions. Significance StatementThe neuromodulators dopamine and norepinephrine modulate a broad range of brain functions, often in concert. One current challenge is to measure dopamine and norepinephrine dynamics simultaneously with high spatial and temporal resolution. We therefore developed a red-dopamine biosensor that has 143-fold higher selectivity for dopamine over norepinephrine. Taking advantage of its high selectivity for dopamine over norepinephrine, this red-dopamine biosensor allowed dual-color fluorescence live imaging for monitoring dopamine and norepinephrine in both HeLa cells and hippocampal neurons in vitro combined with the existing green-norepinephrine biosensor that has 350-fold selectivity for norepinephrine over dopamine. Thus, this approach can provide new opportunities to advance our understanding of high spatial and temporal dynamics of dopamine and norepinephrine in normal and abnormal brain functions.

cell biology↗

bioRxiv2020

Chronic antidepressant treatments rescue reduced REM sleep theta power in a rat social defeat stress model of depression

Although It is widely recognized that virtually all antidepressants (ADs) suppress rapid eye movement (REM) sleep, the effects of chronic AD treatments on sleep abnormalities, especially those of REM sleep, have rarely been investigated comprehensively in animal models of major depressive disorder (MDD). Here, we show that chronic social defeat stress (SDS) in rats induces prolonged social avoidance and MDD-like alterations in sleep architecture (increased REM sleep durations, bouts, and shortened REM latency) even a month after the last SDS. These abnormalities were associated with changes in electroencephalography (EEG) spectra powers, such as reduced REM sleep theta powers during the light phase. Chronic AD treatments significantly ameliorated these behavioral, sleep, and EEG abnormalities, although in some cases not to control levels. Interestingly, the social interaction ratios a month after the last SDS most strongly correlated with the REM sleep theta powers. These results suggest that chronic AD treatments suppress REM sleep durations and bouts as observed in human MDD patients, but, at the same time, increase REM sleep theta power. The latter is an EEG parameter that has never been directly investigated in humans, and which may be responsible for the therapeutic effects of ADs. Significance StatementDisturbed sleep is one of nine diagnostic criteria for major depressive disorder (MDD), which usually resolves with adequate treatment. However, little is known about how antidepressants (AD) work in MDD animal models. Here, we developed a novel rat social defeat stress model demonstrating long-lasting MDD-like sleep disturbances. We found that chronic AD treatments suppressed REM durations and bouts as reported previously, but, at the same time, increased REM sleep theta power. Interestingly, among several sleep parameters, only REM sleep theta power strongly correlated with depressive symptoms at 1M. Thus, REM sleep theta power, an EEG parameter that has never been investigated directly in humans, could be a novel indicator for MDD and/or AD effects.

neuroscience↗

bioRxiv2020

Improvement of phycocyanobilin synthesis for genetically encoded phytochrome-based optogenetics.

Optogenetics is a powerful technique using photoresponsive proteins, and light-inducible dimerization (LID) systems, an optogenetic tool, allow to manipulate intracellular signaling pathways. One of the red/far-red responsive LID system, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phytochromobilin or phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (synPCB), and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated PhyB-PIF and synPCB into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

synthetic biology↗

bioRxiv2019

RaptRanker: in silico RNA aptamer selection from HT-SELEX experiment based on local sequence and structure information

Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.

bioinformatics↗

bioRxiv2019

ERK-mediated mechanochemical waves direct collective cell polarization

During collective migration of epithelial cells, the migration direction is aligned over a large, tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback in individual cells yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and the ERK activation triggers cell contraction. In addition, the contraction of the activated cell pulls neighboring cells via cell-cell junctions, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear cell polarization guarantees unidirectional propagation of ERK activation waves, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration.

cell biology↗

bioRxiv2019

Hierarchical modeling of mechano-chemical dynamics of epithelial sheets across cells and tissue

Collective cell migration is a macroscopic population-level phenomenon that has emerged across hierarchy from mi-croscopic interactions between cells; however, the underlying mechanism remains unclear. Here, we targeted epithe-lial collective cell migration, driven by the mechanical force regulated by chemical signals of traveling ERK activation waves, observed in wound healing. We propose a hierarchical mathematical framework for understanding how cells are orchestrated through mechanochemical cell-cell interaction. We mathematically transformed a particle-based model at the cellular level into a continuum model at the tissue level. The continuum model described relationships between cell migration and mechanochemical variables, namely, ERK activity gradients, cell density, and velocity field, which could be compared with live-cell imaging data. The continuum model recapitulated the ERK wave-induced collective cell migration in wound healing. We also numerically confirmed a consistency between the two models. This framework allows us to connect hierarchical causality from the single-cell level to the tissue level.

biophysics↗

bioRxiv2019

CDCP1 controls compensatory renal growth by integrating Src and Met signaling

Hepatocyte growth factor (HGF) controls various biological responses, including morphogenesis, organ regeneration, and cancer invasion, by activating its receptor, Met. However, the mechanisms that precisely control diverse Met signaling remain unclear. Here, we identified CUB domain-containing protein 1 (CDCP1) as a crucial element of HGF signaling. In MDCK cysts, HGF induced Src activation via CDCP1 upregulation, and CDCP1 ablation abrogated HGF responses, i.e., extended invasive cell protrusions and promoted cell growth/proliferation. Mechanistically, CDCP1 upregulation promoted Src recruitment into lipid rafts to activate Met-associated STAT3. In breast cancer cells in which Met and CDCP1 were co-upregulated, CDCP1 knockdown suppressed HGF-induced cell invasion. Furthermore, in vivo analysis showed that CDCP1 ablation suppressed compensatory renal growth by attenuating Met-STAT3 signaling. These findings demonstrate that CDCP1 plays a crucial role in controlling HGF responses by integrating Src and Met-STAT3 signaling, and provide new insights into the regulatory mechanisms underlying the multifaceted functions of HGF.

cell biology↗