Natural Killer Cell Signal Integration Balances Synapse Symmetry and Migration

Imaging immune surveillance by natural killer (NK) cells has revealed that integration of activating and inhibitory signals determines whether or not NK cells stop to kill the target cell or retain a migratory configuration

Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body

Ebola virus inclusion bodies are liquid organelles whose formation is facilitated by nucleoprotein oligomerization

ABSTRACTViral RNA synthesis of several non-segmented, negative-sense RNA viruses (NNSVs) takes place in inclusion bodies (IBs) that show properties of liquid organelles, which are formed by liquid–liquid phase separation of scaffold proteins. It is believed that this is driven by intrinsically disordered regions (IDRs) and/or multiple copies of interaction domains, which for NNSVs are usually located in their nucleo – and phosphoproteins. In contrast to other NNSVs, the Ebola virus (EBOV) nucleoprotein NP alone is sufficient to form IBs without the need for a phosphoprotein, and to facilitate the recruitment of other viral proteins into these structures. While it has been proposed that also EBOV IBs are liquid organelles, this has so far not been formally demonstrated. Here we used a combination of live cell microscopy, fluorescence recovery after photobleaching assays, and mutagenesis approaches together with reverse genetics-based generation of recombinant viruses to study the formation of EBOV IBs. Our results demonstrate that EBOV IBs are indeed liquid organelles, and that oligomerization but not IDRs of the EBOV nucleoprotein plays a key role in their formation. Additionally, VP35 (often considered the phosphoprotein-equivalent of EBOV) is not essential for IB formation, but alters their liquid behaviour. These findings define the molecular mechanism for the formation of EBOV IBs, which play a central role in the life cycle of this deadly virus

Rab27a and MyoVa are the primary MIph interactors regulating melanosome transport in melanocytes

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plusend binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect MlpMyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.publishersversionpublishe

Semiconductor-to-Insulator Transition in Inter-Electrode Bridge-like Ensembles of Anatase Nanoparticles under a Long-Term Action of the Direct Current

The results of experimental studies of ohmic conductivity degradation in the ensembles of nanostructured anatase bridges under a long-term effect of direct current are presented. Stochastic sets of partially conducting inter-electrode bridges consisting of close-packed anatase nanoparticles were formed by means of the seeding particles from drying aqueous suspensions on the surfaces of silica substrates with interdigital platinum electrodes. Multiple-run experiments conducted at room temperature have shown that ohmic conductivity degradation in these systems is irreversible. It is presumably due to the accumulated capture of conduction electrons by deep traps in anatase nanoparticles. The scaling analysis of voltage drops across the samples at the final stage of degradation gives a critical exponent for ohmic conductivity as ≈1.597. This value satisfactorily agrees with the reported model data for percolation systems. At an early stage of degradation, the spectral density of conduction current fluctuations observed within the frequency range of 0.01–1 Hz decreases approximately as 1/ω, while near the percolation threshold, the decreasing trend changes to ≈1/ω2. This transition is interpreted in terms of the increasing contribution of blockages and subsequent avalanche-like breakdowns of part of the local conduction channels in the bridges into electron transport near the percolation threshold

Normality sensing licenses local T cells for innate-like tissue surveillance

The increasing implication of lymphocytes in general physiology and immune surveillance outside of infection poses the question of how their antigen receptors might be involved. Here, we show that macromolecular aggregates of intraepidermal γδ T cell antigen receptors (TCRs) in the mouse skin aligned with and depended on Skint1, a butyrophilin-like (BTNL) protein expressed by differentiated keratinocytes (KCs) at steady state. Interruption of TCR-mediated ‘normality sensing’ had no impact on γδ T cell numbers but altered their signature phenotype, while the epidermal barrier function was compromised. In addition to the regulation of steady-state physiology, normality sensing licensed intraepidermal T cells to respond rapidly to subsequent tissue perturbation by using innate tumor necrosis factor (TNF) superfamily receptors. Thus, interfering with Skint1-dependent interactions between local γδ T cells and KCs at steady state increased the susceptibility to ultraviolet B radiation (UVR)-induced DNA damage and inflammation, two cancer-disposing factors

Hybrid Perovskite Terahertz Photoconductive Antenna

Hybrid organic–inorganic perovskites, while well examined for photovoltaic applications, remain almost completely unexplored in the terahertz (THz) range. These low-cost hybrid materials are extremely attractive for THz applications because their optoelectronic properties can be chemically engineered with relative ease. Here, we experimentally demonstrate the first attempt to apply solution-processed polycrystalline films of hybrid perovskites for the development of photoconductive terahertz emitters. By using the widely studied methylammonium-based perovskites MAPbI3 and MAPbBr3, we fabricate and characterize large-aperture photoconductive antennas. The work presented here examines polycrystalline perovskite films excited both above and below the bandgap, as well as the scaling of THz emission with the applied bias field and the optical excitation fluence. The combination of ultrafast time-resolved spectroscopy and terahertz emission experiments allows us to determine the still-debated room temperature carrier lifetime and mobility of charge carriers in halide perovskites using an alternative noninvasive method. Our results demonstrate the applicability of hybrid perovskites for the development of scalable THz photoconductive devices, making these materials competitive with conventional semiconductors for THz emission

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

<div><p>Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF₄^- and S1-ADP-BeF_x that mimic S1 ATPase intermediate states S1**-ADP-P_i and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF₄^- and S1-ADP-BeF_x significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.</p></div