Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling.
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We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments. (+info)
The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species.
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Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm. (+info)
Kinesin-5 motors are required for organization of spindle microtubules in Silvetia compressa zygotes.
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BACKGROUND: Monastrol, a chemical inhibitor specific to the Kinesin-5 family of motor proteins, was used to examine the functional roles of Kinesin-5 proteins during the first, asymmetric cell division cycle in the brown alga Silvetia compressa. RESULTS: Monastrol treatment had no effect on developing zygotes prior to entry into mitosis. After mitosis entry, monastrol treatment led to formation of monasters and cell cycle arrest in a dose dependent fashion. These findings indicate that Kinesin-5 motors maintain spindle bipolarity, and are consistent with reports in animal cells. At low drug concentrations that permitted cell division, spindle position was highly displaced from normal, resulting in abnormal division planes. Strikingly, application of monastrol also led to formation of numerous cytasters throughout the cytoplasm and multipolar spindles, uncovering a novel effect of monastrol treatment not observed in animal cells. CONCLUSION: We postulate that monastrol treatment causes spindle poles to break apart forming cytasters, some of which capture chromosomes and become supernumerary spindle poles. Thus, in addition to maintaining spindle bipolarity, Kinesin-5 members in S. compressa likely organize microtubules at spindle poles. To our knowledge, this is the first functional characterization of the Kinesin-5 family in stramenopiles. (+info)
Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules.
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Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules. (+info)
Microtubule dynamics in relation to osmotic stress-induced ABA accumulation in Zea mays roots.
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Microtubules play important roles in many physiological processes such as plant responses to drought stress. Abscisic acid (ABA) accumulates significantly in plants in response to drought conditions, which has been considered as a major response for plants to enhance drought tolerance. In this work, the focus was on the possible roles of microtubules in the induction of ABA biosynthesis in the roots of Zea mays when subjected to osmotic stress. The dynamic changes of microtubules in response to the stress were investigated by immunofluorescence staining, enzyme-linked immunosorbent assay, and a pharmacological approach. Disruption and stabilization of microtubules both significantly stimulated ABA accumulation in maize root cells, although this stimulation was markedly lower than that caused by osmotic stress. Cells in which the microtubule stability had been changed did not respond further to osmotic stress in terms of ABA biosynthesis. However, treatment with both a microtubule de-stabilizer and a stabilizer enhanced the sensitivity of cells to osmotic stress in terms of ABA accumulation. It is suggested that both osmotic stress and changes in microtubule dynamics would trigger maize root cells to biosynthesize ABA, and interactions between osmotic stress and microtubule dynamics would have an effect on ABA accumulation in root cells, although the exact mechanism is not clear at present. (+info)
Rationale and design of a randomized trial to assess the effects of diuretics in heart failure: Japanese Multicenter Evaluation of Long- vs Short-Acting Diuretics in Congestive Heart Failure (J-MELODIC).
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BACKGROUND: Diuretics are the most prescribed medication for heart failure (HF) patients, but clinical evidence of the long-term effects of diuretics are lacking. The present study was designed to compare the therapeutic effects of furosemide, a short-acting loop diuretic, and azosemide, a long-acting one, in patients with HF to test the hypothesis that long-acting diuretics are superior therapy. METHODS AND RESULTS: The Japanese Multicenter Evaluation of LOng- vs short-acting Diuretics In Congestive heart failure (J-MELODIC) is a multicenter, prospective, randomized trial enrolling a total of 300 patients (150 patients in each group). The primary outcome is a composite of cardiovascular death and unplanned admission to hospital for congestive HF. Other outcomes include all-cause mortality, worsening of the symptoms of HF, or a need for modification of therapy. Serial assessment of echocardiographic and neurohumoral parameters will be conducted over a minimum follow-up period of 2 years. CONCLUSIONS: The study results will provide important evidence for the treatment of chronic HF. (+info)
Synthesis, biological evaluation, and molecular modeling of 3,5-substituted-N1-phenyl-N4,N4-di-n-butylsulfanilamides as antikinetoplastid antimicrotubule agents.
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Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leishmania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and methyl ketone moieties. Dicyano compound 5 displays IC(50) values that are comparable to 2b against purified leishmanial tubulin assembly (6.6 vs 7.4 microM), Trypanosoma brucei brucei growth in vitro (0.26 vs 0.18 microM), Leishmania donovani axenic amastigote growth in vitro (4.4 vs 2.3 microM), and in vitro toxicity against Vero cells (16 vs 9.7 microM). Computational studies provide a rationale for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions of the sulfanilamide ring. (+info)
A chemogenomic screening of sulfanilamide-hypersensitive Saccharomyces cerevisiae mutants uncovers ABZ2, the gene encoding a fungal aminodeoxychorismate lyase.
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Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity. (+info)