Microtubules regulate disassembly of epithelial apical junctions

BACKGROUND: Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. RESULTS: Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. CONCLUSION: Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins

Supersymmetric null-surfaces

Single trace operators with the large R-charge in supersymmetric Yang-Mills theory correspond to the null-surfaces in $AdS_5\times S^5$. We argue that the moduli space of the null-surfaces is the space of contours in the super-Grassmanian parametrizing the complex $(2|2)$-dimensional subspaces of the complex $(4|4)$-dimensional space. The odd coordinates on this super-Grassmanian correspond to the fermionic degrees of freedom of the superstring.Comment: v4: added a reference to the earlier work; corrected the formula for the stabilizer of the BMN vacuum; added the discussion of the complex structure of the odd coordinates in Section 3.

Myosin II regulates the shape of three-dimensional intestinal epithelial cysts.

The development of luminal organs begins with the formation of spherical cysts composed of a single layer of epithelial cells. Using a model three-dimensional cell culture, this study examines the role of a cytoskeletal motor, myosin II, in cyst formation. Caco-2 and SK-CO15 intestinal epithelial cells were embedded into Matrigel, and myosin II was inhibited by blebbistatin or siRNA-mediated knockdown. Whereas control cells formed spherical cysts with a smooth surface, inhibition of myosin II induced the outgrowth of F-actin-rich surface protrusions. The development of these protrusions was abrogated after inhibition of F-actin polymerization or of phospholipase C (PLC) activity, as well as after overexpression of a dominant-negative ADF/cofilin. Surface protrusions were enriched in microtubules and their formation was prevented by microtubule depolymerization. Myosin II inhibition caused a loss of peripheral F-actin bundles and a submembranous extension of cortical microtubules. Our findings suggest that inhibition of myosin II eliminates the cortical F-actin barrier, allowing microtubules to reach and activate PLC at the plasma membrane. PLC-dependent stimulation of ADF/cofilin creates actin-filament barbed ends and promotes the outgrowth of F-actin-rich protrusions. We conclude that myosin II regulates the spherical shape of epithelial cysts by controlling actin polymerization at the cyst surface

Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2

Aberrant mechanotransduction and compromised epithelial barrier function are associated with numerous human pathologies including inflammatory skin disorders. However, the cytoskeletal mechanisms regulating inflammatory responses in the epidermis are not well understood. Here we addressed this question by inducing a psoriatic phenotype in human keratinocytes and reconstructed human epidermis using a cytokine stimulation model. We show that the inflammation upregulates the Rho-myosin II pathway and destabilizes adherens junctions (AJs) promoting YAP nuclear entry. The integrity of cell-cell adhesion but not the myosin II contractilityper seis the determinative factor for the YAP regulation in epidermal keratinocytes. The inflammation-induced disruption of AJs, increased paracellular permeability, and YAP nuclear translocation are regulated by ROCK2, independently from myosin II activation. Using a specific inhibitor KD025, we show that ROCK2 executes its effects via cytoskeletal and transcription-dependent mechanisms to shape the inflammatory response in the epidermis

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.Peer reviewe

CAPP Axion Search Experiments with Quantum Noise Limited Amplifiers

The axion is expected to solve the strong CP problem of quantum chromodynamics and is one of the leading candidates for dark matter. CAPP in South Korea has several axion search experiments based on cavity haloscopes in the frequency range of 1-6 GHz. The main effort focuses on operation of the experiments with the highest possible sensitivity. It requires maintenance of the haloscopes at the lowest physical temperature in the range of mK and usage of low noise components to amplify the weak axion signal. We report development and operation of low noise amplifiers for 5 haloscope experiments targeting at different frequency ranges. The amplifiers show noise temperatures approaching the quantum limit.Comment: 6 pages, 7 figures, 29th International Conference on Low Temperature Physics, August 18-24, 2022, Sapporo, Japa

Four-channel System for Characterization of Josephson Parametric Amplifiers

The axion search experiments based on haloscopes at the Center for Axion and Precision Physics Research (CAPP) of the Institute for Basic Science (IBS) in South Korea are performed in the frequency range from 1 GHz to 6 GHz. In order to perform the experiments in a strong magnetic field of 12 T and a large-volume cavity of close to 40 liters, we use He wet dilution refrigerators with immersed superconducting magnets. The measurements require continuous operation for months without interruptions for microwave component replacements. This is achieved by using different cryogenic engineering approaches including microwave RF-switching. The critical components, defining the scanning rate and the sensitivity of the setup, are the Josephson parametric amplifiers (JPA) and cryogenic low noise amplifiers (cLNA) based on high-electron-mobility-transistor (HEMT) technology. It is desirable for both devices to have a wide frequency range and low noise close to the quantum limit for the JPA. In this paper, we show a recent design of a 4-channel measurement setup for JPA and HEMT measurements. The setup is based on a 4-channel wideband noise source (NS) and is used for both JPA and HEMT gain and noise measurements. The setup is placed at 20 mK inside the dry dilution refrigerator. The NS is thermally decoupled from the environment using plastic spacers, superconducting wires and superconducting coaxial cables. We show the gain and noise temperature curves measured for 4 HEMT amplifiers and 2 JPAs in one cool-downComment: to be published in JPS Conference Proceedings (LT29

Josephson Parametric Amplifier in Axion Experiments

The axion is a hypothetical particle, a promising candidate for dark matter, and a solution to the strong CP problem. Axion haloscope search experiments deal with a signal power comparable to noise uncertainty at millikelvin temperature. We use a flux-driven Josephson parametric amplifier (JPA) with the aim of approaching a noise level near the theoretically allowed limit of half quanta. In our measurements to characterize the JPA we have found the added noise to the system with a JPA as the first-stage amplifier to be lower than 110 mK at the frequencies from 0.938 GHz to 0.963 GHz.Comment: to be published in JPS Conference Proceedings (LT29

Cisplatin binding sites on human albumin

Reactions of cisplatin (cis -[PtCl2(NH3)2]) with albumin are thought to play an important role in the metabolism of this anticancer drug. They are investigated here via (i) labeling of cisplatin with 15N and use of two-dimensional 1H,15N NMR spectroscopy, (ii) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (iii) chemical modification of Cys, Met, and His residues, (iv) reactions of bound platinum with thiourea, and (v) gel filtration chromatography. In contrast to previous reports, it is shown that the major sulfur-containing binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Additional monofunctional adducts involving other Met residues and Cys-34 are also observed. During the later stages of reactions of cisplatin with albumin, release of NH3 occurs due to the strong trans influence of Met sulfur, which weakens the Pt-NH3 bonds, and protein cross-linking is observed. The consequences of these findings for the biological activity of cisplatin-albumin complexes are discussed

Activation of Epidermal Toll-Like Receptor 2 Enhances Tight Junction Function: Implications for Atopic Dermatitis and Skin Barrier Repair

Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus skin infections. S. aureus is sensed by many pattern recognition receptors, including Toll-like receptor 2 (TLR2). We hypothesized that an effective innate immune response will include skin barrier repair, and that this response is impaired in AD subjects. S. aureus–derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, claudin-1 (CLDN1), claudin-23 (CLDN23), occludin, and Zonulae occludens 1 (ZO-1) in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape stripping. Tlr2−/− mice had a delayed and incomplete barrier recovery following tape stripping. AD subjects had reduced epidermal TLR2 expression as compared with nonatopic subjects, which inversely correlated (r=-0.654, P=0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may have a role in their incompetent skin barrier