Carbon nanotube recognition by human Siglec-14 provokes inflammation.

For the design and development of innovative carbon nanotube (CNT)-based tools and applications, an understanding of the molecular interactions between CNTs and biological systems is essential. In this study, a three-dimensional protein-structure-based in silico screen identified the paired immune receptors, sialic acid immunoglobulin-like binding lectin-5 (Siglec-5) and Siglec-14, as CNT-recognizing receptors. Molecular dynamics simulations showed the spatiotemporally stable association of aromatic residues on the extracellular loop of Siglec-5 with CNTs. Siglec-14 mediated spleen tyrosine kinase (Syk)-dependent phagocytosis of multiwalled CNTs and the subsequent secretion of interleukin-1ß from human monocytes. Ectopic in vivo expression of human Siglec-14 on mouse alveolar macrophages resulted in enhanced recognition of multiwalled CNTs and exacerbated pulmonary inflammation. Furthermore, fostamatinib, a Syk inhibitor, blocked Siglec-14-mediated proinflammatory responses. These results indicate that Siglec-14 is a human activating receptor recognizing CNTs and that blockade of Siglec-14 and the Syk pathway may overcome CNT-induced inflammation.

Tim4, a macrophage receptor for apoptotic cells, binds polystyrene microplastics via aromatic-aromatic interactions.

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1ß secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases.

Torque generating properties of Tetrahymena ciliary three-headed outer-arm dynein.

Eukaryotic cilia/flagella are cellular bio-machines that drive the movement of microorganisms. Molecular motor axonemal dyneins in the axoneme, which consist of an 9 + 2 arrangement of microtubules, play an essential role in ciliary beating. Some axonemal dyneins have been shown to generate torque coupled with the longitudinal motility of microtubules across an array of dyneins fixed to the coverglass surface, resulting in a corkscrew-like translocation of microtubules. In this study, we performed three-dimensional tracking of a microbead coated with axonemal outer-arm dyneins on a freely suspended microtubule. We found that microbeads coated with multiple outer-arm dyneins exhibited continuous right-handed helical trajectories around the microtubule. This unidirectional helical motion differs from that of other types of cytoplasmic dyneins, which exhibit bidirectional helical motility. We also found that, in an in vitro microtubule gliding assay, gliding microtubules driven by outer-arm dyneins tend to turn to the left, causing a curved path, suggesting that the outer-arm dynein itself is able to rotate on its own axis. Two types of torque generated by the axonemal dyneins, corresponding to the forces used to rotate the microtubule unidirectionally with respect to the long and short axes, may regulate ciliary beating with complex waveforms.

Shaping of T Cell Functions by Trogocytosis.

Trogocytosis is an active process whereby plasma membrane proteins are transferred from one cell to the other cell in a cell-cell contact-dependent manner. Since the discovery of the intercellular transfer of major histocompatibility complex (MHC) molecules in the 1970s, trogocytosis of MHC molecules between various immune cells has been frequently observed. For instance, antigen-presenting cells (APCs) acquire MHC class I (MHCI) from allografts, tumors, and virally infected cells, and these APCs are subsequently able to prime CD8+ T cells without antigen processing via the preformed antigen-MHCI complexes, in a process called cross-dressing. T cells also acquire MHC molecules from APCs or other target cells via the immunological synapse formed at the cell-cell contact area, and this phenomenon impacts T cell activation. Compared with naïve and effector T cells, T regulatory cells have increased trogocytosis activity in order to remove MHC class II and costimulatory molecules from APCs, resulting in the induction of tolerance. Accumulating evidence suggests that trogocytosis shapes T cell functions in cancer, transplantation, and during microbial infections. In this review, we focus on T cell trogocytosis and the related inflammatory diseases.

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

Circular orientation fluorescence emitter imaging (COFEI) of rotational motion of motor proteins.

Single-molecule fluorescence polarization technique has been utilized to detect structural changes in biomolecules and intermolecular interactions. Here we developed a single-molecule fluorescence polarization measurement system, named circular orientation fluorescence emitter imaging (COFEI), in which a ring pattern of an acquired fluorescent image (COFEI image) represents an orientation of a polarization and a polarization factor. Rotation and pattern change of the COFEI image allow us to find changes in the polarization by eye and further values of the parameters of a polarization are determined by simple image analysis with high accuracy. We validated its potential applications of COFEI by three assays: 1) Detection of stepwise rotation of F1-ATPase via single quantum nanorod attached to the rotary shaft γ; 2) Visualization of binding of fluorescent ATP analog to the catalytic subunit in F1-ATPase; and 3) Association and dissociation of one head of dimeric kinesin-1 on the microtubule during its processive movement through single bifunctional fluorescent probes attached to the head. These results indicate that the COFEI provides us the advantages of the user-friendly measurement system and persuasive data presentations.

Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain.

The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, ß, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule.

Torque generation by axonemal outer-arm dynein.

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αßγ) as well as proteolytically generated two-headed (ßγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αßγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed ßγ did not display this sensitivity. In the assays on lawns containing mixtures of α and ßγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αßγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

Low-dose mithramycin exerts its anticancer effect via the p53 signaling pathway and synergizes with nutlin-3 in gynecologic cancers.

MDM2 is a direct negative regulator of p53. The p53-independent mdm2-P1 and p53-dependent mdm2-P2 promoters have been recently shown to harbor Sp1 binding sites. Mithramycin, an inhibitor of Sp1 DNA binding, has been used clinically to treat hypercalcemia and some types of neoplastic disorders. In this study, we investigated the mechanisms behind the anticancer effect of mithramycin. In gynecologic cancer cells expressing wild-type p53, mithramycin stabilized p53 and increased the expression of the p53 downstream target genes PUMA and p21, arrested the cell cycle, and induced apoptosis. This activation of the p53 signaling pathway was a specific effect of MTH at concentrations <50 nm. Mithramycin temporally decreased transcription of both the mdm2-P1 and -P2 promoters. This was followed by a subsequent increase of mdm2-P2 promoter activity by activated p53. Up-regulated MDM2 was in its active form, and consequently attenuated p53 activity. Although mithramycin activated p53 and suppressed the growth of human gynecologic cancer cell xenografts in mice, this was accompanied with a secondary up-regulation of MDM2. Combined treatment with mithramycin and nutlin-3, a drug that inhibits MDM2-p53 interaction, overcame a secondary up-regulation of MDM2 and synergistically inhibited cancer cell growth by inducing apoptosis through activation of the p53 signaling pathway. These observations provide a better understanding of the mechanisms of mithramycin activity, and suggest a potential role for combining mithramycin and nutlin-3 as a chemotherapeutic treatment for gynecologic cancers.

Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.

p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.

Surface observation for seed-mediated growth attachment of gold nanoparticles on a glassy carbon substrate.

A seed-mediated growth method for surface modification was applied to the attachment of gold nanoparticles (AuNPs) to glassy carbon (GC) surfaces. By simply immersing a GC plate at first into a seed solution containing 4 nm Au nano-seed particles and then into a growth solution containing HAuCl(4), ascorbic acid and cetyltrimethyammonium bromide, AuNPs could be successfully attached to the GC surface via the growth of nanoparticles. A possible control of the size and density of AuNPs on GC was examined by observing surface images with a field-emission scanning electron microscope (FE-SEM) after several preparations with different immersion times. Compared with previous results on the growth of AuNPs on indium tin oxide (ITO) surfaces, it was characteristic that the AuNPs attached to GC surfaces exhibited smaller size and higher density as well as a flatter and non-crystal-like morphology. In addition, for performing the dense attachment of regular nano-sized AuNPs on GC surfaces, immersion for 2 h into the growth solution was sufficient. Longer immersion for 24 h caused an irregular growth of bold Au micro-crystals, while 24 h was necessary in the case of AuNPs on ITO surfaces. Shorter seeding and growth times were found to be effective for a sparse attachment of smaller Au nanoparticles whose size was ca. 20 nm. It was clarified that the seed-mediated growth method for surface modification was valid for fabricating a nanointerface composed of AuNPs on GC surfaces.

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster.

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

Analysis of the modification site in a small molecule-modified peptide by ion trap/time-of-flight hybrid mass spectrometry.

Ion trap/time-of-flight hybrid mass spectrometers are powerful tools for the detailed structural analysis of modified peptides. We have analyzed Met-Lys-bradykinin modified with deoxycholate at the amino-terminus or the epsilon-amino group as model peptides. These two modified peptides produced fragment ions with the same nominal but different exact masses in tandem mass spectrometry with low-energy collision-induced dissociation. Accurate high-resolution analysis coupled with MS(3) allowed us to distinguish the deoxycholate modification sites in the modified peptides.

Molecular mechanisms underlying dexamethasone inhibition of iNOS expression and activity in C6 glioma cells.

The synthetic glucocorticoid dexamethasone is routinely used to stabilize patients with malignant gliomas. One putative target for glucocorticoid action is inducible nitric oxide synthase (iNOS), which is produced by the tumor cells as well as the host immune cells. In this study, we characterize the stimulatory effects of lipopolysaccharide (LPS) and the cytokine, tumor necrosis factor-alpha (TNFalpha), as well as the inhibitory effect of glucocorticoids, on iNOS gene expression and activity in C6 glioma cells cultured in vitro. LPS significantly increased iNOS mRNA expression, peaking at 6 h, while nitrite formation increased with time up to 72 h. Although TNFalpha alone induced neither iNOS mRNA expression nor nitrite formation, it significantly potentiated the effect of LPS on both. iNOS activity induced by LPS with or without TNFalpha was dose-dependently inhibited by dexamethasone, reaching a maximum of approximately 83% inhibition. This was completely reversed by the addition of RU38486, an antagonist of glucocorticoid receptors (GR). Dexamethasone inhibited iNOS mRNA expression; however, the maximum inhibition obtained was only 10%. These results suggest that as for induction of iNOS activity in C6 cells in vitro, the stimulatory effect of LPS is mainly due to an action at the transcriptional level. TNFalpha does not have intrinsic inducing activity, but has potentiating effects at the transcriptional and possibly at the posttranscriptional levels in the presence of LPS. The inhibitory effect of dexamethasone is GR-mediated and is mainly due to action at the posttranscriptional level.