Biology subjects

Yanagisawa, H.

Publications and source records attributed to Yanagisawa, H..

33 records · Page 2Linked to original sources

bioRxiv2023

Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 [A], we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 [A]. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

biochemistry↗

bioRxiv2023

Distinct roles of α- and β-tubulin C-terminal tails for ciliary function as revealed by a CRISPR/Cas9 mediated gene editing in Chlamydomonas

- and {beta}-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular alga Chlamydomonas possesses only two -tubulin genes and two {beta}-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the -tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the {beta}-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the {beta}-tubulin CTT. Therefore, our study reveals distinct and important roles of - and {beta}-tubulin CTT in the formation and function of cilia. Summary statementChlamydomonas mutants were produced by CRISPR/Cas9 mediated gene editing to investigate ciliary function of tubulin C-terminal tails (CTTs). We found that - and {beta}-tubulin CTTs are essential for ciliary motility and assembly.

cell biology↗

bioRxiv2022

Parallel cryo electron tomography on in situ lamellae

In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.

biophysics↗

bioRxiv2022

The extracellular matrix fibulin 7 maintains epidermal stem cell heterogeneity during skin aging

Tissue stem cells divide infrequently as a protective mechanism against internal and external stresses associated with aging. Here, we demonstrate that slow- and fast-cycling interfollicular epidermal stem cells in mouse skin undergo distinct aging processes. Two years of lineage tracing reveals that Dlx1+ slow-cycling clones expanded into the fast-cycling stem cell territory, while the number of Slc1a3+ fast-cycling clones gradually declined. Transcriptome analysis further indicated that the molecular properties of each stem cell population are altered with age. Mice lacking fibulin 7, an extracellular matrix (ECM), show early impairments resembling epidermal stem cell aging, such as the loss of fast-cycling clones, delayed wound healing, and increased expression of inflammation- and differentiation-related genes. Fibulin 7 interacts with structural ECM and matricellular proteins, and the overexpression of fibulin 7 in primary keratinocytes results in slower proliferation in the absence or presence of inflammatory cytokine IL-6. Thus, these results suggest that fibulin 7 plays a crucial role in maintaining tissue resilience and epidermal stem cell heterogeneity during skin aging.

developmental biology↗

bioRxiv2022

Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 [A] resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

immunology↗

bioRxiv2021

Partial endothelial-to-mesenchymal transition (EndMT) contributes to lumen re-organization after carotid arteryligation

Endothelial-to-mesenchymal transition (EndMT) is a fundamental process in vascular remodeling. Carotid artery ligation is commonly used for induction of neointima formation and vessel stenosis; however, the precise regulatory mechanism of vascular remodeling is not entirely understood. In this study, we showed that resident endothelial cells (ECs) are the origin of neointima cells and ECs transiently expressed CD45 in the early stage of neointima formation accompanied by increased expression of EndMT markers. In vitro, CD45-positive EndMT was induced by stabilization of HIF-1 with cobalt chloride or VHL inhibitor in human primary ECs, which mimicked the hypoxic condition of ligated artery, and promoted the formation of integrin 11-SHARPIN complex. Notably, a CD45 phosphatase inhibitor disrupted this complex, thereby destabilizing cell-cell junctions. These results suggest that the CD45 activity is required for the retention of an EC phenotype and cell-cell junctions during EndMT (termed "partial EndMT"). We thus propose a novel mechanism of partial EndMT that contributes to lumen re-organization during vascular injury.

molecular biology↗

bioRxiv2021

On-site genomic epidemiological analysis of antimicrobial-resistant bacteria in Cambodia with portable laboratory equipment

The rapid emergence of carbapenemase-producing Gram-negative bacteria (CPGNB) is a global threat due to the high mortality of infection and limited treatment options. Although there have been many reports of CPGNB isolated from Southeast Asian countries, to date there has been no genetic analysis of CPGNB isolated from Cambodia. Sequence-based molecular epidemiological analysis enables a better understanding of the genotypic characteristics and epidemiological significance of antimicrobial-resistant (AMR) bacteria in each country, and allows countries to enact measures related to AMR issues. In this study, we performed on-site genomic epidemiological analysis of CPGNB isolated in Cambodia using a portable laboratory equipment called Bento Lab, which combines a PCR thermal cycler, microcentrifuge, gel electrophoresis apparatus, and LED transilluminator, along with the MinION nanopore sequencer. PCR targeting of major carbapenemase genes using Bento Lab revealed that two Escherichia coli isolates and one Acinetobacter baumannii isolate harbored carbapenemase genes: blaNDM, blaOXA-48, and blaOXA-23, respectively. The results of phenotypic diagnostic tests for CPGNB, such as the carbapenem inactivation method and double-disk diffusion test using a specific inhibitor of metallo-{beta}-lactamases, were consistent with their AMR genotypes. Whole-genome sequencing analysis using MinION suggested that blaNDM-5 and blaOXA-181 genes were carried on plasmids (93.9-kb plasmid with IncFIA, IncFIB, IncFII, and IncQ1 replicons, and 51.5-kb plasmid with the IncX3 replicon, respectively) in E. coli, and that blaOXA-23 was encoded in two locations on the chromosome of A. baumannii. Plasmids carrying blaNDM-5 or blaOXA-181 in E. coli were highly structurally identical to plasmids prevalent in Enterobacterales in China and other countries, suggesting that they disseminated from a common evolutionary origin. Our findings demonstrate the potential impact of portable laboratory equipment on AMR bacteria research in hospitals and research centers with limited research facilities, and provide the first glimpse into the genomic epidemiology of CPGNB in Cambodia.

microbiology↗

bioRxiv2020

Modelling acceptable novelty transitions with emotional habituation: Effects of uncertainty and prediction error on preference changes

Previous mathematical models developed to optimize the degree of novelty in product design have represented novelty by emotional dimensions such as arousal (surprise) and valence (positivity and negativity). Formalizing arousal as Bayesian information gain and valence as a function of arousal based on Berlynes arousal potential theory, the model indicates that novelty becomes acceptable with repeated exposure and changes the preferences of the users. Hence, acceptable novelty transitions are important in the design of long-term product experience. We propose a mathematical model of acceptable novelty transitions with emotional habituation based on the emotional dimension model. We formalized valence as a function of information-theoretic free-energy and expressed free-energy as a function of three parameters: initial prediction error, initial uncertainty, and noise of sensory stimulus. To verify whether the transition speed of acceptable novelty depends on the initial uncertainty in our model, we analysed the responses of participants to historic artworks; we manipulated the uncertainty level by varying the obscurity of the presented pieces and the prediction error by rendering them in different artistic styles. We used the subjective reports of valence in response to the samples as measures of valence levels. The experimental results support our hypothesis.

neuroscience↗

bioRxiv2020

How predictability affects habituation to novelty?

One becomes accustomed to repeated exposures, even for a novel event. In the present study, we investigated how predictability affects habituation to novelty by applying a mathematical model of arousal that we previously developed, and conducted a psychophysiological experiment to test the model prediction. We formalized habituation to novelty as a decrement in Kullback-Leibler divergence from Bayesian prior to posterior (i.e., information gain) representing arousal evoked from a novel event through Bayesian update. The model predicted an interaction effect between initial uncertainty and initial prediction error (i.e., predictability) on habituation to novelty: The greater the initial uncertainty, the faster the information gain decreases (i.e., the sooner one is habituated). Experimental results using subjective reports of surprise and event-related potential (P300) evoked by visual-auditory incongruity supported the model prediction. Our findings suggest that in highly uncertain situations, repeated exposure to stimuli may enhance habituation to novel stimuli.

neuroscience↗

bioRxiv2020

Defining compartmentalized stem and progenitor populations with distinct cell division frequency in the ocular surface epithelium

SummaryAdult tissues contain label-retaining cell (LRC)s, which are relatively slow-cycling and considered to represent a unique property of tissue stem cell (SC)s. In the ocular surface epithelium, LRCs are detected in the limbus, a boundary between cornea and conjunctiva, and the fornix of the conjunctiva; however, the character of LRCs and identity of SCs remain unclear due to lack of appropriate molecular markers. Here we show that the ocular surface epithelium accommodates spatially distinct stem/progenitor populations with different cell division frequency. By combining EdU pulse-chase analysis and lineage tracing with three CreER transgenic mouse lines: Slc1a3CreER, Dlx1CreER and K14CreER, we detect distinct dynamics of epithelial SCs in the cornea and conjunctiva. In the limbus, long-lived SCs are labeled with Slc1a3CreER and they either migrate centripetally toward the central cornea or laterally expand their clones within the limbal region. In the central cornea, cells are mostly non-LRCs, labeled by Dlx1CreER and K14CreER, and the number of clones declines after a short period of time with rare long-lasting clones, suggesting their properties as short-lived progenitor cells. In the conjunctival epithelium, which consists of bulbar, fornix and palpebral conjunctiva, each territory is regenerated by compartmentalized, distinct SC populations without migrating one region to another. The severe damage of the cornea leads to the cancellation of SC compartments, causing conjunctivalization of the eye, whereas milder limbal injury induces a rapid increase of laterally-expanding clones in the limbus. Taken together, our work provides lineage tracing tools of the eye and defines compartmentalized, multiple SC/progenitor populations in homeostasis and their behavioral changes in response to injury.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Cryo-electron tomography of cardiac myofibrils reveals a contraction-induced lattice twist in the Z-discs.

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by -actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has been elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actinactinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the -actinin showed a contraction-induced swing motion along with a global twist in the actin lattice. Our observation suggests that the elasticity and the integrity of the Z-disc during the muscle contraction cycle are maintained by the structural flexibility within the actin-actinin complex.

biochemistry↗

bioRxiv2020

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

biochemistry↗

bioRxiv2019

Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP signaling pathway in the remodeling of blood vessels

RationaleThe extracellular matrix (ECM) initiates mechanical cues and transduces intracellular signaling through matrix-cell interactions. In the blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from ECM and how they coordinate with a mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood.\n\nObjectiveThe aim of this study was to elucidate the crucial mediator(s) and molecular signaling pathway(s) involved in matrix mechanotransduction during remodeling of the vessel wall.\n\nMethods and ResultsWe performed secretome analysis using rat vascular smooth muscle cells (SMCs) under cyclic stretch and examined matrix-cell interactions and cell behavior. We found that the matricellular protein thrombospondin-1 (Thbs1) was secreted upon cyclic stretch and bound to integrin v{beta}1, thereby recruiting vinculin and establishing focal adhesions. RNA-sequence (RNA-seq) analysis revealed that deletion of Thbs1 in vitro markedly affected the target gene expression of Yes-associated protein (YAP). Consistently, we found that Thbs1 promotes nuclear shuttling of YAP in response to cyclic stretch, which depends on the small GTPase Rap2 and Hippo pathway, and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload by transverse aortic constriction (TAC), whereas it suppressed neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall.\n\nConclusionsThbs1 serves as a mechanical stress-triggered extracellular mediator of mechanotransduction that acts via integrin v{beta}1 to establish focal adhesions and promotes nuclear shuttling of YAP. We thus propose a novel mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.\n\nSubject codesO_LIVascular Disease\nC_LIO_LIGenetically Altered and Transgenic Models\nC_LIO_LIVascular Biology\nC_LIO_LICell Signaling/Signal Transduction\nC_LI

molecular biology↗

bioRxiv2018

Inner lumen proteins stabilize doublet microtubules in cilia/flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

cell biology↗

bioRxiv2018

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

molecular biology↗