Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.The direction of cell expansion is important for determining the shape of whole plant body. Cortical microtubules are assumed to be responsible for anisotropic expansion of plant cells (Wasteneys and Galway, 2003; Lloyd and Chan, 2004; Mathur, 2004; Baskin, 2005; Paredez et al., 2008). The prevailing view is that cortical microtubule arrays direct or constrain the movement of the cellulose synthase complexes and thus align nascent cellulose microfibrils in the same direction in the innermost layer of the cell wall (Baskin, 2001), although some other mechanisms may also be involved (Baskin, 2001; Sugimoto et al., 2003; Wasteneys, 2004).It is evident that orientation of cortical microtubules plays an essential role in creating the distinct shape of higher plant organs, even if there is uncertainty over the mechanism by which microtubules influence morphogenesis. The importance of cortical microtubule arrays for anisotropic growth has been documented by pharmacological studies and experiments with helical growth mutants of Arabidopsis (Arabidopsis thaliana). Mutants on α- and β-tubulins as well as microtubule-associated proteins show either left-handed or right-handed helical growth (Thitamadee et al., 2002; Nakajima et al., 2004; Sedbrook et al., 2004; Shoji et al., 2004). The rapidly elongating cells of these mutants skew consistently either to the right or to the left and exhibit cortical microtubule arrays that form shallow helices with fixed handedness (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). Cortical microtubule arrays in the left-handed helical growth mutants form right-handed helix, whereas those in right-handed helical growth mutants form left-handed helix (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). These results indicate that dysfunctional cortical microtubules are arranged in helical arrays and affect the direction of cell expansion.The gravitational force is one of the environmental factors that determine the plant body shape. Under hypergravity conditions produced by centrifugation, plants generally have a shorter and thicker body (Soga et al., 2006). Namely, hypergravity modifies growth anisotropy. In Arabidopsis hypocotyls, the expression of most α- and β-tubulin genes was up-regulated by hypergravity (Yoshioka et al., 2003; Matsumoto et al., 2007). In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical microtubules into parallel arrays (Skagen and Iversen, 1999), and in azuki bean (Vigna angularis) epicotyls it increased the percentage of cells with longitudinal cortical microtubules (Soga et al., 2006). The reorientation of cortical microtubules from transverse to longitudinal directions may be involved in modifications by hypergravity to growth anisotropy.The aim of this study was to clarify the roles of cortical microtubules in gravity-induced modifications to development of stem organs. For this purpose, we examined the changes in growth, morphology, and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis amino acid substitution mutants in α-tubulin structure, tua3, tua4, and tua6, grown under 1g and 300g conditions. We have reported the possible involvement of mechanosensitive ion channels (mechanoreceptors) in hypergravity-induced modifications to growth and cell wall properties (Soga et al., 2004, 2005, 2006). Thus, we also examined the effect of blockers of mechanoreceptors on helical growth and orientation of cortical microtubule arrays in the tubulin mutants.     

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.     

Tight relationship between two photosystems is robust in rice leaves under various nitrogen conditions

Journal of Plant Research - Leaf nitrogen (N) level affects not only photosynthetic CO2 assimilation, but also two photosystems of the photosynthetic electron transport. The quantum yield of...     

A binocular model for the simple cell

We authors propose a mathematical model for simple cell binocular response. It comprises two Gabor-type receptive fields (RF) having the same RF center, preferred spatial frequency, and preferred orientation. The model integrates the equally weighted signals from both eyes and performs a threshold operation. Poggio and Fischer (1977) classified binocular disparity cells in the striate cortex into four groups: tuned excitatory (TE), tuned inhibitory (TI), near, and far cells. They also found that most of the TE cells are ocularly balanced and that the other three types are usually unbalanced. This model can imitate these four types of disparity sensitivities and their ocular dominance tendency. We perform model fittings to Poggio's data using the “simulated annealing” method and discuss parameter dependence of the model's response. The model can also respond with exceptional disparity sensitivity: i.e., flat type, alternating type, and intermediate type.