Programmed cell death 1 (PD-1) is highly expressed on exhausted T cells and inhibits activation. Antibodies that block the interaction between PD-1 its ligand prevent this inhibitory signal reverse dysfunction, providing beneficial anti-tumor responses in a substantial number of patients. Mechanisms for induction maintenance high expression have not been fully understood. Utilizing genome-wide loss-of-function screening method based CRISPR-Cas9 system, we identified genes involved core...

The acquired immune system, mainly composed of T and B lymphocytes, plays a key role in protecting the host from infection. It is important technically challenging to identify cell types their activation status living intact cells, without staining or killing cells. Using Raman spectroscopy, we succeeded discriminating between cells visualized labeling. Although spectra were similar, they could be distinguished by discriminant analysis principal components. activated with anti-CD3 anti-CD28...

Abstract Fluorescent proteins have been widely used in biology because of their compatibility and varied applications living specimens. are often undesirably sensitive to intracellular conditions such as pH ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence yellow fluorescent protein (YFP) depends on concentration solution dependence be enhanced by...

<title>Abstract</title> Various improvements have been made to chimeric antigen receptor (CAR)-T cells enhance their antitumor effects and expand indications various cancer types. Although second-generation CARs directly induce second signals in a linear pathway via own costimulatory domain such as CD28, CAR-T themselves express receptors, which additional parallel. To clarify the differences between these two types of signals, we analyzed CD28-mediated signalosomes using high-resolution...

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance damaged and aged red blood cells (RBCs) from circulation. While a number previous experimental studies have observed changes distribution after oxidative treatment, details how these clusters are formed their properties change under different conditions remained poorly understood. To address issues, framework that enables simultaneous monitoring temporal spatial following oxidation is...

Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) already been recognized as the next efficacious treatment, but there is still limited understanding mechanism LAG-3-mediated suppression. Here, utilizing high-resolution molecular imaging, we find a CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting...

The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in determination characteristics cancer cells, including chemotherapy sensitivity. Resistance to is often linked dysfunction tumor suppressor p53; however, it remains elusive whether ECM microenvironment interferes with p53 activation cells. Here, we show that, MCF-7 breast stiffness influences induced by antitumor drug doxorubicin. Cell growth inhibition doxorubicin was increased response rigidity a...

The coinhibitory receptor, PD-1, is of major importance for the suppression T cell activation in various types immune responses. A high-resolution imaging study showed that PD-1 forms a signalosome, "PD-1 microcluster", with phosphatase, SHP2, to dephosphorylate TCR/CD3 complex and its downstream signaling molecules. Such consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 expressed professional antigen-presenting cells also some tumor cells, which possibly explains...

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular is shown to function areas without internal actomyosin contractility, sensing force at leading edge cell migration. Circular dichroism shows CasSD intrinsically disordered with dominant polyproline type II conformations. Strongly conserved placental mammals, sequence exhibits a...

Abstract Using Raman spectral imaging, we visualized the cell state transition during differentiation and constructed hypothetical potential landscapes for attractors of cellular states on a space composed parameters related to shape spectra. As models differentiation, used myogenic C2C12 line mouse embryonic stem cells. imaging can validate amounts locations multiple components that describe such as proteins, nucleic acids lipids; thus, it report single cell. Herein, using nuclei in...

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix. Tyrosine phosphorylation of focal proteins is crucial for cells communicate with environment. However, mechanisms that transmit actin cytoskeletal motion environment drive cell migration poorly understood. We find movement p130Cas (Cas), a mechanosensor at adhesions, correlates retrograde flow, and depends upon actomyosin contraction Cas substrate domain (CasSD). This indicates...

With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a method to evaluate them. Here we establish an advanced imaging system visualize human PD-1 microclusters, which minimal T cell receptor (TCR) signaling unit co-localizes with inhibitory co-receptor vitro. In these...

We studied the role of Src SH3 domain in its dynamics at cell membrane using site-directed mutagenesis and live imaging. Physiologically, proliferation migration require expression family kinases. Hyperactivation molecules has been detected various cancer cells. Although activation mechanism intensively studied, are still unclear. also exist cellular locations, we found that activated mainly localized peripheral adhesion sites. phosphorylation status subdomain conformations thought to...

ABSTRACT Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported p130Cas supports cell through the binding to Src as well phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts cytoskeletons. observe actin-binding protein, tensin 1, co-localizes Cas, but not its phosphorylation-defective mutant, adhesions leading...

Contractile force generated in actomyosin stress fibers (SFs) is transmitted along SFs to the extracellular matrix (ECM), which contributes cell migration and sensing of ECM rigidity. In this study, we show that efficient transmission relies on actin crosslinking by α-actinin. Upon reduction α-actinin-mediated crosslinks, myosin II activity induced flows filaments SFs, leading a decrease traction exertion ECM. The fluidized maintained their cable integrity probably through enhanced...

Abstract Our current understanding of molecular biology provides a clear picture how the genome, transcriptome and proteome regulate each other, but chemical environment cell plays role in cellular regulation remains much to be studied. Here we show an imaging method using hybrid fluorescence-Raman microscopy that measures micro-environment associated with protein expression patterns living cell. Simultaneous detection fluorescence Raman signals, realised by spectrally separating two modes...

We evaluated usability of a previously developed genetically encoded molecular crowding sensor in various biological phenomena. Molecular refers to intracellular regions that are occupied more by proteins and nucleotides than water molecules is thought have strong effect on protein function. To evaluate crowding, usually the diffusion coefficient probe used because it related mobility surrounding agents. Recently, sensors based Förster resonance energy transfer were reported. In present...

The immune system in tolerance maintains cell diversity without responding to self-antigens. Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) inhibit T-cell activation through various molecular mechanisms. However, several key questions are still not resolved, including how Tregs control the response on basis of their self-skewed receptor repertoire and avoid impeding relevant immunity against pathogens. Here, we show that promote proliferation conventional presence excessive...

Freestanding cell-sheets are valuable bio-materials for use in regenerative medicine and tissue engineering. Because experience various mechanical stimulations during handling, it is important to understand the responses of cells these stimulations. Here, we demonstrate changes localization proteins stretching fibroblast cell-sheets. These known be involved mechano-sensing. Upon stretching, actin filaments appear parallel direction. At cell-cell junctions, β-catenin forms clusters that...

A co-signaling receptor, 2B4, has dual effects in immune cells, but its actual functions T cells remain elusive. Here, using super-resolution imaging technology with an immunological synapse model, we showed that 2B4 forms "2B4 microclusters" immediately after 2B4-CD48 binding. lipid phosphatase, SHIP-1, subsequently combined to form coinhibitory signalosomes, leading the suppression of cytokine production. An activating adapter, SLAM-associated protein (SAP), attenuated clustering SHIP-1...

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal proteins is crucial for cells communicate with environment. However, mechanisms that transmit actin cytoskeletal motion environment drive cell migration poorly understood. We find movement p130Cas (Cas, also known as BCAR1), a mechanosensor at adhesions, correlates retrograde flow and depends upon actomyosin contraction Cas substrate...