Biology subjects

Kimura, A.

Publications and source records attributed to Kimura, A..

29 records · Page 2Linked to original sources

bioRxiv2019

Evaluation of 3D markerless motion capture accuracy using OpenPose with multiple video cameras

There is a need within human movement sciences for a markerless motion capture system, which is easy to use and suffciently accurate to evaluate motor performance. This study aims to develop a 3D markerless motion capture technique, using OpenPose with multiple synchronized video cameras, and examine its accuracy in comparison with optical marker-based motion capture. Participants performed three motor tasks (walking, countermovement jumping, and ball throwing), with these movements measured using both marker-based optical motion capture and OpenPose-based markerless motion capture. The differences in corresponding joint positions, estimated from the two different methods throughout the analysis, were presented as a mean absolute error (MAE). The results demonstrated that, qualitatively, 3D pose estimation using markerless motion capture could correctly reproduce the movements of participants. Quantitatively, of all the mean absolute errors calculated, approximately 47% were less than 20 mm and 80% were less than 30 mm. However, 10% were greater than 40 mm. The primary reason for mean absolute errors exceeding 40mm was that OpenPose failed to track the participants pose in 2D images owing to failures, such as recognition of an object as a human body segment, or replacing one segment with another depending on the image of each frame. In conclusion, this study demonstrates that, if an algorithm that corrects all apparently wrong tracking can be incorporated into the system, OpenPose-based markerless motion capture can be used for human movement science with an accuracy of 30mm or less.

neuroscience↗

bioRxiv2019

Elasticity and Topography-Controlled Collagen Hydrogels Mimicking Native Cellular Milieus

Accumulating evidence demonstrates that the elasticity and topography of a cell culture substrate influence cell behavior, in addition to its chemical composition. However, cellular responses to in vivo extracellular matrix (ECM), a hydrogel of proteins (mainly collagen) with various elasticity and a nanometer-to micrometer-scale topography, remain to be elucidated owing to a lack of substrate that provides such complex cues. This study introduces novel collagen hydrogels that can combine, for the first time, elastic, topographic, and compositional cues that recapitulate native ECM. A simple and reagent-free method based on radiation crosslinking alters ECM-derived collagen solutions into hydrogels with a well-defined and tunable elastic modulus covering the broad range of soft tissues (1-236 kPa) and microtopographies while ensuring intrinsic biological functionality of collagen. These collagen hydrogels enabled investigating cell responses to soft topographic cues such as those encountered in vivo, revealing that topography overrides the elasticity and structurally constrains cell morphology by controlling actin cytoskeleton organization. The collagen hydrogels not only reduce in vivo and in vitro behavioral disparity of cells by mimicking native ECM but also facilitate the design of artificial ECM to control cell function and fate in tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2019

Pivotal involvement of the CX3CL1-CX3CR1 axis for the recruitment of M2-TAMs in skin carcinogenesis

We previously revealed the crucial roles of CX3CL1-CX3CR1 axis in skin wound healing. Although repeated wounds frequently develop into skin cancer, the roles of CX3CL1 in skin carcinogenesis remain elusive. Here, we proved that CX3CL1 protein expression and CX3CR1+ macrophages were observed in human skin cancer tissues. Similarly, we observed the enhancement of CX3CL1 expression and the abundant accumulation of CX3CR1+ tumor-associated macrophages (TAMs) with M2 phenotypes in the skin carcinogenesis process induced by the combined treatment with 7,12-dimethylbenz-(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). In this mouse skin carcinogenesis process, CX3CR1+ TAMs exhibited M2 phenotypes with the expression of Wnt3a and angiogenic molecules including vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9. Compared to wild-type mice, CX3CR1-deficient mice showed fewer numbers of skin tumors with a lower incidence. Concomitantly, M2-macrophage numbers and neovascularization reduced with the depressed expression of angiogenic factors and Wnt3a. Thus, the CX3CL1-CX3CR1 axis can crucially contribute to skin carcinogenesis by regulating the accumulation and functions of TAMs. Thus, this axis can be a good target for preventing and/or treating skin cancers.

cancer biology↗

bioRxiv2019

Perturbation of the titin/MURF1 signaling complex is associated with hypertrophic cardiomyopathy in a fish model and in human patients.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of the patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line-A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found in both medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM.

developmental biology↗

bioRxiv2019

Multiplexed 129Xe HyperCEST MRI detection of genetically-reconstituted bacterial protein nanoparticles in human cancer cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically-encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4, and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive and genetically-encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

biophysics↗

bioRxiv2019

Cold Vacuum Extracts of Double Cherry Blossom (Gosen-Sakura) Leaves Show Antitumor Activity

The present research examines the possibility of finding bio-molecular compounds from the double cherry blossom (termed as Gosen-Sakura of Gosen-city, Niigata-prefecture, Japan) leaves, which have been long used in the preparation of the traditional Japanese sweet (wagashi) - sakura-mochi. Based on its indicated anti-microbial properties historically, our study provides a new low temperature vacuum extraction method for extracting near natural form of water soluble leaf (cell) extracts from the Gosen-Sakura, and demonstrates the presence of some novel compound(s) with anti-tumor cell lines proliferation inhibitory affects through the MTT assay. To our knowledge, no reports exist on the sakura tree leaf (cell) extracts inhibiting tumor cell line growth. We further examined and compared the effects of known compounds with anti-tumor activity, coumarin and benzyl alcohol with Gosen-Sakura leaf extract; results lead us to hypothesize that the Gosen-Sakura leaf extract contains substance(s) other than the above 2 known compounds, with antitumor effect. Additionally, we speculate on the underlying mechanism of action of the Gosen-Sakura leaf extract by targeting cell division at the point of DNA synthesis and causing apoptosis. In conclusion, we present scientific evidence on the presence of a certain novel biomolecule(s), with anti-tumor activity, in the Gosen-Sakura leaf which has been long used as a Japanese - the sakura-mochi.

plant biology↗

bioRxiv2018

Choice between 1- and 2-furrow cytokinesis in Caenorhabditis elegans embryos with tripolar spindles

Excess numbers of centrosomes often lead to multipolar spindles, and thus probably to multipolar mitosis and aneuploidy. In Caenorhabditis elegans, approximately 70% of the paternal emb-27APC6 mutant embryonic cells contained more than 2 centrosomes and formed multipolar spindles. However, only 30% of the cells with tripolar spindles formed 2 cytokinetic furrows. The rest formed 1 furrow, like normal cells. To investigate the mechanism how the cells avoided to form 2 cytokinetic furrows even with a tripolar spindle, we conducted live-cell imaging in emb-27APC6 mutant cells. We found that the chromatids were aligned only on 2 of the 3 sides of the tripolar spindle, and the angle of the tripolar spindle relative to the long axis of the cell correlated with the number of cytokinetic furrow. Our numerical modeling showed that the combination of cell shape, cortical pulling forces, and heterogeneity of centrosome size determines whether cells with tripolar spindle form 1 or 2 cytokinetic furrows.

cell biology↗

bioRxiv2018

Impaired chromosome segregation results in sperms with excess centrosomes in emb-27APC6 mutant C. elegans

The anaphase-promoting complex (APC) is a major regulator of chromosome segregation and is implicated in centriole engagement, whose de-regulation causes abnormal number of centrosomes. The emb-27 gene in C. elegans encodes a subunit of APC. The paternal emb-27 mutant was reported to show cell division with multiple furrows, suggesting the presence of excess centrosomes. In this study, we examined the number of centrosomes and the mechanism underlying de-regulation of centrosome number in emb-27 mutants. Our observations indicated excess centrosomes in emb-27 sperms, which resulted in zygotes with excess centrosomes. Further, the secondary spermatocyte of emb-27 produced reduced number of spermatids, which is likely the direct cause of the excess number of centrosomes per sperm. We propose that a chromosome segregation defect in emb-27 induced centrosome separation defect, resulting in reduced number of buds. Additionally, treatment with cytoskeletal inhibiting drugs indicated presence of three kinds of forces working in combination to move the centrosomes into the spermatids. The present study suggested a novel role of microtubule in the budding cytokinesis of spermatocytes.

cell biology↗

bioRxiv2018

Microinjection into the Caenorhabditis elegans embryo using an uncoated glass needle enables cell lineage visualization and reveals cell-non-autonomous adhesion control

Microinjection is a useful method in cell biology, with which exogenous substances are introduced into a cell in a location- and time-specific manner. The Caenorhabditis elegans embryo is an important model system for cell and developmental biology. Applying microinjection to the C. elegans embryo had been difficult due to the rigid eggshell surrounding the embryo. In 2013, microinjection method using a carbon-coated quartz needle for the C. elegans embryo was reported. To prepare the needle, unfortunately, special equipment is required and thus a limited number of researchers can use this method. In this study, we established a method for the microinjection of drugs, dyes, and microbeads into the C. elegans embryo using an uncoated glass needle that can be produced in a general laboratory. This method enabled us to easily detect cell lineage up to adult stages by injecting a fluorescent dye into a blastomere. We also found a cell-non-autonomous control mechanism of cell adhesion; specifically, the injection of an actin inhibitor into one cell at the 2-cell stage enhanced adhesion between daughter cells of the other cell. Our microinjection method is expected to be used for broad studies and could facilitate various discoveries using C. elegans.

cell biology↗

bioRxiv2017

The tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/{beta}-catenin signaling and accurate chromosome segregation, and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that C. elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par-aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle pulling forces. Our results document a physical basis for spindle-pulling force attenuation, which may be generally used in asymmetric cell division, and when disrupted potentially contributes to division defects in cancer.\n\nSignificance StatementAPC (adenomatous polyposis coli) is a Wnt signaling component as well as a microtubule-associated protein, and its mutations are frequently associated with colorectal cancers in humans. Although APC stabilizes microtubules (MTs), its mechanical role during cell division is largely unknown. Here we show that APC is an attenuator of forces acting on the mitotic spindle during asymmetric cell division of the C. elegans zygote. We performed live-imaging, laser-microsurgery, and numerical simulation to show how APC suppresses spindle pulling force generation by stabilizing microtubule plus-ends and reducing microtubule catastrophe frequency at the cell cortex. Our study is the first to document a mechanical role for the APC protein, and provides a physical basis for spindle-pulling force attenuation.

cell biology↗

bioRxiv2017

An Anisotropic Attraction Model For The Diversity And Robustness Of Cell Arrangement In Nematodes

In early embryogenesis stages in animals, cells are arranged into a species-specific pattern in a robust manner. The cell arrangement patterns are diverse, even among close relatives. We evaluated how the diversity and robustness are achieved in developing embryos. We succeeded in reproducing different patterns of cell arrangements observed in various nematode species using Caenorhabditis elegans embryos by changing the eggshell shapes. This implies that the diversity of cell arrangements can be explained by differences in a shape parameter. Additionally, we found that the cell arrangement was robust against eggshell deformation. Computational modeling revealed that, in addition to repulsion forces, attraction forces are sufficient for this robustness. Genetic perturbation experiments demonstrated that attraction forces derived from cell adhesion are necessary for the robustness. The proposed model accounts for both diversity and robustness of cell arrangements and contributes to our understanding of how diversity and robustness are achieved in developing embryos.

cell biology↗