Biology subjects

Deguchi, S.

Publications and source records attributed to Deguchi, S..

34 records · Page 2Linked to original sources

bioRxiv2023

AP2A1 is upregulated upon replicative senescence of human fibroblasts to strengthen focal adhesions via integrin β1 translocation along stress fibers

Aging proceeds with accumulation of senescent cells in multiple organs. Senescent cells become large in size compared to young cells, which promotes further senescence and age-related diseases. Currently, the molecular mechanism behind the maintenance of such huge cell architecture undergoing senescence remains poorly understood. Here we focus on reorganization of actin stress fibers induced upon replicative senescence of human fibroblasts, typically used as a senescent cell model. We identified, together with our previous proteomic study, that AP2A1 (alpha 1 adaptin subunit of the adaptor protein 2) is upregulated in senescent cells along the length of stress fibers, which are enlarged following the increase in the whole cell size. We then revealed that knockdown of AP2A1 in senescent cells suppresses key senescence-associated phenotypes, which include decreased cell area and lowered expression of major senescence markers. Meanwhile, AP2A1 overexpression in young cells induced the opposite effects that rather advance senescence, suggesting that AP2A1 may be used as a senescence marker. We found that AP2A1 is colocalized with integrin {beta}1, and both of them move linearly along stress fibers. We further observed that focal adhesions are enlarged in senescent cells to reinforce cell adhesions to the substrate. These results suggest that senescent cells maintain their large size by strengthening the anchorage to the substrate by supplying integrin {beta}1 via translocation along stress fibers. This mechanism may work efficiently in senescent cells, compared with a case relying on random diffusion of integrin {beta}1, given the enlarged cell size and resulting increase in travel time and distance for endocytosed vesicle transportation.

cell biology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 XBB.1.5 variant

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.

microbiology↗

bioRxiv2023

Genetic and functional diversity of beta-N-acetylgalactosamine residue-targeting glycosidases expanded by deep-sea metagenome

{beta}-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. {beta}-N-Acetylgalactosaminidases hydrolyze {beta}-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-{beta}-N-acetylgalactosaminidases that specifically act on {beta}-N-acetylgalactosamine residues, has been documented so far. In this study, we identified three novel glycoside hydrolase families distributed among all three domains of life and characterized eight novel {beta}-N-acetylgalactosaminidases and {beta}-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and diversify their substrate specificities (endo-, dual-endo/exo-, and exo-) through the accumulation of mutations and insertional amino acid sequences. The diverse {beta}-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present novel evolutionary pathways for expanding their substrate specificity.

biochemistry↗

bioRxiv2023

Knowledge "installed" diffusion model predicts the geometry of actin cytoskeleton from cell morphology

Cells exhibit various morphological characteristics due to their physiological activities, and changes in cell morphology are inherently accompanied by the assembly and disassembly of the actin cytoskeleton. Stress fibers are a prominent component of the actin-based intracellular structure and are highly involved in numerous physiological processes, e.g., mechanotransduction and maintenance of cell morphology. Although it is widely accepted that variations in cell geometry interact with the distribution and localization of stress fibers, it remains unclear if there are underlying geometric principles between the cell morphology and actin cytoskeleton. Here we present a machine learning system, which uses the diffusion model, that can convert the cell shape to the distribution of stress fibers. By training with corresponding datasets of cell shape and stress fibers, our system learns the conversion to generate the stress fiber images from its corresponding cell shape. The predicted stress fiber distribution has good agreement with the experimental data, and the overlap region of predicted and experimentally observed stress fibers reaches 79.3 {+/-}12.4%. We then found some unknown natures such as a linear relation relationship between the stress fiber length and cell area. With this "installed" conversion relation between cellular morphology and corresponding stress fibers localization, our system could perform virtual experiments that provide a visual map showing the probability of stress fiber distribution from the virtual cell shape. Our system provides a powerful approach to seek further hidden geometric principles between the cell morphologies and actin cytoskeletons.

bioinformatics↗

bioRxiv2022

A model explaining environmental stiffness-dependent migration of fibroblasts with a focus on maturation of intracellular structures

Cell migration is fundamental to many biological processes, while it remains elusive how cells modulate their migration upon different environmental stiffness. In this work, we focus on the structural maturity of actin stress fibers to explain the substrate stiffness-dependent emergence of different cell migration velocity. We demonstrate that fibroblasts migrate longer distances on softer elastic substrates, and the distance is increased by lowering the myosin-driven contractile force. Stress fibers, the major intracellular structure to generate and sustain contractile forces, were found to be less mature in structure on soft substrate than on stiff substrate. Based on these experimental results, we present a minimal mathematical model to capture the salient features of how the substrate stiffness alters the migration velocity. Specifically, the ability of cells to generate large contractile forces is limited on soft substrate according to the Hookes law. The inverse relationship between the cellular force and migration velocity is described by the Hills muscle equation. These mathematical descriptions suggest that the migration velocity is raised on softer substrate where cells exert a lower magnitude of contractile forces. Cells undergoing faster movement make stress fibers less mature in structure as mathematically described by the maturation model, thereby limiting the ability to sustain the force and in turn allowing for consistent increase in cell migration velocity on soft substrate again according to the Hookes law and Hills muscle equation, respectively. Thus, our model, reproducing the basic trend of the experimental results, provides insights into the mechanisms of environmental cue-dependent migratory behavior of cells.

biophysics↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

microbiology↗

bioRxiv2022

Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.

microbiology↗

bioRxiv2022

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75

SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

microbiology↗

bioRxiv2022

Emergence of multiple set-points of cellular homeostatic tension

Stress fibers (SFs), a contractile actin bundle in nonmuscle mesenchymal cells, are known to intrinsically sustain a constant level of tension or tensional stress, a process called cellular tensional homeostasis. Malfunction in this homeostatic process has been implicated in many diseases such atherosclerosis, but its mechanisms remain incompletely understood. Interestingly, the homeostatic stress in individual SFs is altered upon recruitment of -smooth muscle actin in particular cellular contexts to reinforce the preexisting SFs. While this transition of the set-point stress is somewhat a universal process observed across different cell types, no clear explanation has been provided as to why cells end up possessing different stable stresses. To address the underlying physics, here we describe that imposing a realistic assumption on the nature of SFs yields the presence of multiple set-points of the homeostatic stress, which transition among them depending on the magnitude of the cellular tension. We derive non-dimensional parameters that characterize the extent of the transition and predict that SFs tend to acquire secondary stable stresses if they are subject to as large a change in stiffness as possible or to as immediate a transition as possible upon increasing the tension. This is a minimal and simple explanation, but given the frequent emergence of force-dependent transformation of various subcellular structures in addition to that of SFs, the theoretical concept presented here would offer an essential guide to addressing potential common mechanisms governing complicated cellular mechanobiological responses.

biophysics↗

bioRxiv2022

Analysis of chemical and mechanical behaviors in living cells by continuum mechanics-based FRAP

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently-labeled beta-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate of beta-actin was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the strain rate along stress fibers but, interestingly, not with the absolute strain magnitude. Moreover, stress fibers are subjected to centripetal flow as well as both contractile and tensile strains along them. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

biophysics↗

bioRxiv2022

A model for determining the length distribution of actin filaments in cells

Tensional homeostasis is a cellular process whereby nonmuscle cells such as fibroblasts keep a constant level of intracellular tension and signaling activities. Cells are allowed thanks to tensional homeostasis to adapt to mechanical stress, but the detailed mechanism remains unclear. Here we address from a theoretical point of view what is required for maintaining cellular tensional homeostasis. A constrained optimization problem is formulated to analytically determine the probability function of the length of individual actin filaments (AFs) responsible for sustaining cellular tension. An objective function composed of two entropic quantities measuring the extent of formation and dispersion of AFs within cells is optimized under two constraint functions dictating a constant amount of actin molecules and tension that are arguably the two most salient features of tensional homeostasis. We then derive a specific probability function of AFs that is qualitatively consistent with previous experimental observations, in which short AF populations preferably appear. Regarding the underlying mechanism, our analyses suggest that the constraint for keeping the constant tension level makes long AF populations smaller in number because long AFs have a higher chance to be involved in bearing larger forces. The specific length distribution of AFs is thus required for achieving the constrained objectives, by which individual cells are endowed with the ability to stably maintain a homeostatic tension throughout the cell, thereby potentially allowing cells to locally detect deviation in the tension, keep resulting biological functions, and hence enable subsequent adaptation to mechanical stress. Although minimal essential factors are included given the actual complexity of cells, our approach would provide a theoretical basis for understanding complicated homeostatic and adaptive behavior of the cell.

biophysics↗

bioRxiv2021

Long-term molecular turnover of actin stress fibers revealed by advection-reaction analysis in fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) is a versatile technique to evaluate the intracellular molecular exchange called turnover. Physicochemical models of FRAP typically consider the molecular diffusion and chemical reaction that simultaneously occur on a time scale of seconds to minutes. Particularly for long-term measurements, however, an advection effect can no longer be ignored, which transports the proteins in specific directions within the cells and accordingly shifts the spatial distribution of the local chemical equilibrium. Nevertheless, existing FRAP models have not considered the spatial shift, and as such, the turnover rate is often analyzed without considering the spatiotemporally updated chemical equilibrium. Here we develop a new FRAP model aimed at long-term measurements to quantitatively determine the two distinct effects of the advection and chemical reaction, i.e., the different major sources of the change in fluorescence intensity. To validate this approach, we carried out FRAP experiments on actin in stress fibers over a time period of more than 900 s, and the advection rate was shown to be comparable in magnitude to the chemical dissociation rate. We further found that the actin-myosin interaction and actin polymerization differently affect the advection and chemical dissociation. Our results thus suggest that the distinction between the two effects is indispensable to extract the intrinsic chemical properties of the actin cytoskeleton from the observations of complicated turnover in cells.

biophysics↗

bioRxiv2021

Proteome of actin stress fibers

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from huma fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 263 proteins are associated with SFs, and 101 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

cell biology↗

bioRxiv2021

Machine learning based cellular contractile force detection: a new approach to predict cell mechanics by images

Combining experiments with artificial intelligence algorithms, we propose a new machine learning based approach to extract the cellular force distributions from the microscope images. The full process can be divided into three steps. First, we culture the cells on a special substrate allowing to measure both the cellular traction force on the substrate and the corresponding substrate wrinkles simultaneously. The cellular forces are obtained using the traction force microscopy (TFM), at the same time that cell-generated contractile forces wrinkle their underlying substrate. Second, the wrinkle positions are extracted from the microscope images. Third, we train the machine learning system with GAN (generative adversarial network) by using sets of corresponding two images, the traction field and the input images (raw microscope images or extracted wrinkle images), as the training data. The network understands the way to convert the input images of the substrate wrinkles to the traction distribution from the training. After sufficient training, the network is utilized to predict the cellular forces just from the input images. Our system provides a powerful tool to evaluate the cellular forces efficiently because the forces can be predicted just by observing the cells under the microscope, which is a way simpler method compared to the TFM experiment. Additionally, the machine learning based approach presented here has the profound potential for being applied to diverse cellular assays for studying mechanobiology of cells. Significance StatementCell-generated forces are indispensable determinants of fundamental cell functions such as motility and cell division. As such, quantifying how the forces change upon perturbations to the cells such as gene mutations and drug administration is of profound importance. Here we present a novel machine learning based system that allows for efficient estimations of the forces that are determined only by "observing" microscope images. Given that the cellular traction forces are regulated downstream of diverse signaling pathways, our system - that helps significantly improve the throughput of the measurements - presents a new, high throughput platform for real time analysis of the effects of a massive number of genetic and molecular perturbations on the forces and resulting cell mechanics.

biophysics↗

bioRxiv2020

Statistical profiling reveals correlations between the cell response to and structural sequence of Rho-GAPs

Rho-GTPase-activating proteins (Rho-GAPs) are essential upstream regulators of the Rho family of GTPases. Currently, it remains unclear if the phenotypic change caused by perturbations to a Rho-GAP is predictable from its structural sequence. Here we analyze the relationship between the morphological response of cells to the silencing of Rho-GAPs and their primary structure. For all possible pairs of 57 different Rho-GAPs expressed in MCF10A epithelial cells, the similarity in the Rho-GAP silencing-induced morphological change was quantified and compared to the similarity in the primary structure of the corresponding pairs. We found a distinct correlation between the morphological and structural similarities in a specific group of RhoA-targeting Rho-GAPs. Thus, the family-wide analysis revealed a common feature shared by the specific Rho-GAPs.

cell biology↗

bioRxiv2020

Determining the domain-level reaction-diffusion properties of cellular proteins by incorporating genetic engineering to FRAP-based framework

Proteins in cells undergo repeated association to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical reaction, it is difficult to attribute the measured quantities to the nature of specific domains of the probed proteins particularly if, as is often the case, the protein has multiple domains to independently interact with the same types but different molecules. Motivated by the common goal in cell signaling research aimed at identifying the protein domains responsible for particular intermolecular interactions, here we describe a new approach to determining the domain-level reaction and pure diffusion properties. To validate this methodology, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created with genetic engineering, and the difference among the mutants in FRAP response is analyzed. We demonstrate that transgelin-2 in cells interacts with F-actin via two separate domains, and the chemical equilibrium constant of the interaction is determined at the individual domain levels. Its pure diffusion properties independent of the association to F-actin is also obtained. This approach requires some effort to construct the mutants, but instead enables in situ domain-level determination of the physicochemical properties, which will be useful, as specifically shown here for transgelin-2, in addressing the signaling mechanism of cellular proteins.

bioengineering↗