Caenorhabditis elegans unc-82 Encodes a Serine/Threonine Kinase Important for Myosin Filament Organization in Muscle During Growth

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cell-shape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome

Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices

Validation of the ICRF antenna coupling code RAPLICASOL against TOPICA and experiments

In this paper we validate the finite element code RAPLICASOL, which models radiofrequency wave propagation in edge plasmas near ICRF antennas, against calculations with the TOPICA code. We compare the output of both codes for the ASDEX Upgrade 2-strap antenna, and for a 4-strap WEST-like antenna. Although RAPLICASOL requires considerably fewer computational resources than TOPICA, we find that the predicted quantities of experimental interest (including reflection coefficients, coupling resistances, S- and Z-matrix entries, optimal matching settings, and even radiofrequency electric fields) are in good agreement provided we are careful to use the same geometry in both codes

A locked mode indicator for disruption prediction on JET and ASDEX upgrade

The aim of this paper is to present a signal processing algorithm that, applied to the raw Locked Mode signal, allows us to obtain a disruption indicator in principle exploitable on different tokamaks. A common definition of such an indicator for different machines would facilitate the development of portable systems for disruption prediction, which is becoming of increasingly importance for the next tokamak generations. Moreover, the indicator allows us to overcome some intrinsic problems in the diagnostic system such as drift and offset. The behavior of the proposed indicator as disruption predictor, based on crossing optimized thresholds of the signal amplitude, has been analyzed using data of both JET and ASDEX Upgrade experiments. A thorough analysis of the disruption prediction performance shows how the indicator is able to recover some missed and tardy detections of the raw signal. Moreover, it intervenes and corrects premature or even wrong alarms due to, e.g., drifts and/or offsets

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present