Drosophila melanogaster γ-TuRC is dispensable for targeting γ-tubulin to the centrosome and microtubule nucleation

In metazoans, γ-tubulin acts within two main complexes, γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs). In higher eukaryotes, it is assumed that microtubule nucleation at the centrosome depends on γ-TuRCs, but the role of γ-TuRC components remains undefined

Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis

The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle

Nitrogen-mediated metabolic patterns of susceptibility to Botrytis cinerea infection in tomato (Solanum lycopersicum) stems

International audienceSevere N stress allows an accumulation of C-based compounds but impedes that of N-based compounds required to lower the susceptibility of tomato stem to Botrytis cinerea . Abstract Botrytis cinerea , a necrotrophic filamentous fungus, forms potentially lethal lesions on the stems of infected plants. Contrasted levels of susceptibility to B. cinerea were obtained in a tomato cultivar grown on a range of nitrate concentration: low N supply resulted in high susceptibility while high N supply conferred a strong resistance. Metabolic deviations and physiological traits resulting from both infection and nitrogen limitation were investigated in the symptomless stem tissue surrounding the necrotic lesion. Prior to infection, nitrogen-deficient plants showed reduced levels of nitrogen-based compounds such as amino acids, proteins, and glutathione and elevated levels of carbon-based and defence compounds such as α-tomatine and chlorogenic acid. After B. cinerea inoculation, all plants displayed a few common responses, mainly alanine accumulation and galactinol depletion. The metabolome of resistant plants grown under high N supply showed no significant change after inoculation. On the contrary, the metabolome of susceptible plants grown under low N supply showed massive metabolic adjustments, including changes in central metabolism around glutamate and respiratory pathways, suggesting active resource mobilization and production of energy and reducing power. Redox and defence metabolisms were also stimulated by the infection in plants grown under low N supply; glutathione and chlorogenic acid accumulated, as well as metabolites with more controversial defensive roles, such as polyamines, GABA, branched-chain amino acids and phytosterols. Taken together, the results showed that nitrogen deficiency, although leading to an increase in secondary metabolites even before the pathogen attack, must have compromised the constitutive levels of defence proteins and delayed or attenuated the induced responses. The involvement of galactinol, alanine, cycloartenol and citramalate in the tomato stem response to B. cinerea is reported here for the first time

Alary muscles and TARMs, a novel type of striated muscles maintaining internal organs positions

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Nitrogen-mediated metabolic patterns of susceptibility to Botrytis cinerea infection in tomato (Solanum lycopersicum) stems

International audienceSevere N stress allows an accumulation of C-based compounds but impedes that of N-based compounds required to lower the susceptibility of tomato stem to Botrytis cinerea . Abstract Botrytis cinerea , a necrotrophic filamentous fungus, forms potentially lethal lesions on the stems of infected plants. Contrasted levels of susceptibility to B. cinerea were obtained in a tomato cultivar grown on a range of nitrate concentration: low N supply resulted in high susceptibility while high N supply conferred a strong resistance. Metabolic deviations and physiological traits resulting from both infection and nitrogen limitation were investigated in the symptomless stem tissue surrounding the necrotic lesion. Prior to infection, nitrogen-deficient plants showed reduced levels of nitrogen-based compounds such as amino acids, proteins, and glutathione and elevated levels of carbon-based and defence compounds such as α-tomatine and chlorogenic acid. After B. cinerea inoculation, all plants displayed a few common responses, mainly alanine accumulation and galactinol depletion. The metabolome of resistant plants grown under high N supply showed no significant change after inoculation. On the contrary, the metabolome of susceptible plants grown under low N supply showed massive metabolic adjustments, including changes in central metabolism around glutamate and respiratory pathways, suggesting active resource mobilization and production of energy and reducing power. Redox and defence metabolisms were also stimulated by the infection in plants grown under low N supply; glutathione and chlorogenic acid accumulated, as well as metabolites with more controversial defensive roles, such as polyamines, GABA, branched-chain amino acids and phytosterols. Taken together, the results showed that nitrogen deficiency, although leading to an increase in secondary metabolites even before the pathogen attack, must have compromised the constitutive levels of defence proteins and delayed or attenuated the induced responses. The involvement of galactinol, alanine, cycloartenol and citramalate in the tomato stem response to B. cinerea is reported here for the first time

Augmin is stably associated with spindle microtubules.

<p>(A–D) FRAP of spindle-associated GFP-Dgt2 in wild-type metaphase I oocytes (A), in wild-type prometaphase/metaphase syncytial embryos (B), in oocytes depleted of γ-tubulin37C by RNAi (C), and in <i>ncd^D</i> homozygous mutant oocytes (D). A typical meiotic figure used for FRAP is shown for each. Error bars are SEM. n≥15 in meiosis and n≥11 in mitosis. (E) Western blot of oocytes using an antibody which recognises all γ-tubulin in oocytes in wild type and after depletion of γ-tubulin37C by RNAi. (F) Two hypothetical models for stable association of Augmin with spindle microtubules. Our data are consistent with the “stabilise and then nucleate” model.</p

Chromosomes fail to congress in <i>wac</i> mutant oocytes.

<p>(A) Chromosome movement in wild-type and <i>wacΔ</i> oocytes expressing Rcc1-mCherry. Scale bar = 10 µm. Time = min:sec. (B) The degree of chromosome congression in wild-type and <i>wacΔ</i> oocytes. The spread of the chromosome mass along the spindle axis, excluding the 4^th chromosome (the double arrow in the diagram), in six oocytes each plotted from nuclear envelope breakdown (time 0) over time.</p

Augmin accumulation at spindle poles is correlated with chromosome congression.

<p>(A, B) GFP-Dgt2 and Wac-GFP localise to wild-type acentrosomal spindle poles. (C) Dgt6 localises to spindle poles in wild-type oocytes by immunostaining. (D) The Augmin level in spindle pole regions is well correlated with the level of chromosome congression. Live oocytes expressing GFP-Dgt2 and Rcc1-mCherry were used to measure two parameters: the spread of the chromosome mass (including the 4th chromosomes) along the spindle axis (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003562#pgen-1003562-g001" target="_blank">Figure 1B</a>), and the intensity of GFP-Dgt2 signal above the background (as in Methods & Materials) for each spindle. Correlation between the chromosome spread and the log of GFP-Dgt2 intensity is significant (r = −0.772, p<0.01, n = 26).</p