Biology subjects

Kumamoto, Y.

Publications and source records attributed to Kumamoto, Y..

6 recordsLinked to original sources

bioRxiv2025

Phase-targeting rapid cryofixation of the beating heart and histological analysis unveil contractile state-dependent sarcomere dynamics

The heart is a functional syncytium consisting of numerous cardiomyocytes that repetitively exhibit coordinated contractions/relaxations. However, the extent to which myocyte sarcomere arrangements in the heart differ across beats is unknown. To examine this, we conducted cardiac phase-targeting rapid cryofixation of Langendorff-perfused rat hearts. We adjusted the timepoint of cryogen exposure to the electrically paced heart and observed phase-dependent differences in the sarcomere length (SL) of subepicardial myocytes by -actinin immunohistochemistry, namely a significantly shorter SL during systole than during diastole. We detected spatially inhomogeneous SL distributions by generating a heatmap of the myocardium. For peak systole the SL heatmap exhibited nearly uniform SL shortening within and among the individual myocytes with some myocardia exhibiting nonuniform SLs. During diastole, the heart showed predominant SL elongation, which was also accompanied by patchy distributions of locally short-SL regions, reflecting inhomogeneous SLs. This SL inhomogeneity was attenuated by pharmacological relaxation by 2,3-butanedione monoxime. The heatmap of the rapidly-frozen heart during ventricular fibrillation also revealed inhomogeneous SLs within and among individual myocytes. Overall, cardiac phase-targeting cryofixation unveiled in-depth behaviors on SL in the heart. Our cryofixation strategy will open a new horizon to clarify precise spatiotemporal changes in sarcomere structures and understand cardiac functions.

biophysics↗

bioRxiv2023

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

biophysics↗

bioRxiv2023

Rapid activation of IL-2 receptor signaling by CD301b+DC-derived IL-2 dictates the outcome of helper T cell differentiation

Effector T helper (Th) cell differentiation is fundamental to functional adaptive immunity. Different subsets of dendritic cells (DCs) preferentially induce different types of Th cells, but the fate instruction mechanism for Th type 2 (Th2) differentiation remains enigmatic, as the critical DC-derived cue has not been clearly identified. Here, we show that CD301b+ DCs, a major Th2-inducing DC subset, drive Th2 differentiation through cognate interaction by kick-starting IL-2 receptor signaling in CD4T cells. Mechanistically, CD40 engagement induces IL-2 production selectively from CD301b+ DCs to maximize CD25 expression in CD4 T cells, which is required specifically for the Th2 fate decision. On the other hand, CD25 in CD301b+ DCs facilitates directed action of IL-2 toward cognate CD4T cells. Furthermore, CD301b+ DC-derived IL-2 skews CD4T cells away from the T follicular helper fate. These results highlight the critical role of DC-intrinsic CD40-IL-2 axis in bifurcation of Th cell fate.

immunology↗

bioRxiv2023

Time-deterministic cryo-optical microscopy

We developed a technique to rapidly freeze biological systems during observation under an optical microscope, which allows for detailed optical observation at a precise time in the biological dynamics at high spatial resolution and quantifiability under low-temperature conditions. Our study further found that ion distributions and molecular conformations can be fixed in space and time, demonstrated by visualization of calcium waves rapid-frozen at an instant using fluorescent ion indicators. These results show that time-deterministic cryo-optical microscopy can suspend cellular dynamics while maintaining molecular and ionic states, allowing us to view cellular dynamics in a way that has not been possible before. One-Sentence SummaryRapid freezing of dynamic biological systems during optical observation presents time information to the fixed samples

biophysics↗

bioRxiv2022

A helminth mimic of TGF-β, TGM, enhances regenerative cutaneous wound healing and modulates immune cell recruitment and activation

Intestinal helminth parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response including anti-inflammatory and tissue repair pathways. TGF-{beta} mimic (TGM), an ES molecule secreted by Heligmosomoides polygyrus (Hp), binds TGF-{beta} receptors yet lacks structural homology to TGF-{beta} and exhibits distinct receptor interactions. We demonstrate TGM treatment enhanced wound healing and tissue regeneration in an in vivo wound biopsy model. TGM, in a 1.5% carboxymethylcellulose solution, was topically administered beneath a Tegaderm layer. Through histological analysis, increased restoration of normal tissue structure in the wound beds of TGM-treated mice was observed during mid- to late-stage wound healing. These observations included accelerated re-epithelialization and hair follicle regeneration, without increased scarring. Flow cytometric and gene expression analysis showed differential expansion of myeloid populations at different stages of wound healing. This included enhanced early accumulation and persistence of macrophages in TGM-treated wounds during the initial inflammatory phase. Additionally, the percentage of alternatively activated (M2) macrophages expressing CD206 was reduced with TGM treatment during early and mid-stage wound healing. scRNAseq analysis of TGM-treated wounds indicate upregulation of multiple wound healing-associated genes without expression of CD206 within macrophage subsets. Experiments with truncated TGM constructs revealed that the TGF{beta}-R binding domain was essential in enhancing the wound healing response. In summary, TGM can accelerate skin wound healing and pro-restorative maturation through its interaction with the TGF-{beta} receptor and stimulate the recruitment and reprogramming of specific macrophage subsets. This study indicates a role for TGM as a potential novel therapeutic option for enhanced wound healing. One-Sentence SummaryA helminth-derived protein leads to rapid wound closure, skin regeneration, and reprogramming of macrophage activation through TGF-{beta}R binding.

immunology↗

bioRxiv2020

Optimal CD4T Cell Priming in Lymph Nodes Requires Repertoire Scanning by CD301b+ Migratory cDC2 Cells

Activation of CD4T cells by conventional dendritic cells (cDC) is pivotal in adaptive immunity. However, while the activation mechanism of antigen-specific CD4T cells has been extensively studied, the cellular mechanism that leads to the selection of cognate CD4T cell clones out of the polyclonal pool is incompletely understood. Here, we show that, in the reactive lymph nodes, newly homed naive polyclonal CD4T cells are temporarily retained before leaving the lymph node. This stop-and-go traffic of CD4T cells provides an adequate time window for efficient scanning and timely priming of antigen-specific clones. Mechanistically, upon immunization, CD301b+ DCs, a major subset of migratory cDC2 cells, quickly migrate to the draining lymph node and settle in the areas near the high endothelial venules, where they retain incoming polyclonal CD4T cells through MHCII-dependent but antigen-independent mechanisms while concurrently providing cognate stimuli to prime antigen-specific CD4T cells. These results indicate that CD301b+ DCs function as an immunological "display window" for CD4T cells to efficiently scan their antigen specificity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/276410v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@118d9b7org.highwire.dtl.DTLVardef@7daf2eorg.highwire.dtl.DTLVardef@17808c0org.highwire.dtl.DTLVardef@186780e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINewly homed polyclonal CD4T cells are temporarily retained in the reactive lymph nodes. C_LIO_LIDepletion of CD301b+ DCs results in shorter dwell time of CD4T cells in the draining lymph node and delayed priming of antigen-specific clones. C_LIO_LIThe transient retention of polyclonal CD4T cells in the draining lymph node requires MHCII expression on CD301b+ DCs but not cognate antigen. C_LIO_LICD301b+ DCs are required for robust expansion of rare antigen-specific CD4T cell clones and their skewing toward Th2 cells. C_LI

immunology↗