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

Rapid identification of causal mutations in tomato EMS populations via mapping-by-sequencing

The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6-12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping

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 subunitm 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.</p

Human c-Myc Isoforms Differentially Regulate Cell Growth and Apoptosis in Drosophila melanogaster

The human c-myc proto-oncogene, implicated in the control of many cellular processes including cell growth and apoptosis, encodes three isoforms which differ in their N-terminal region. The functions of these isoforms have never been addressed in vivo. Here, we used Drosophila melanogaster to examine their functions in a fully integrated system. First, we established that the human c-Myc protein can rescue lethal mutations of the Drosophila myc ortholog, dmyc, demonstrating the biological relevance of this model. Then, we characterized a new lethal dmyc insertion allele, which permits expression of human c-Myc in place of dMyc and used it to compare physiological activities of these isoforms in whole-organism rescue, transcription, cell growth, and apoptosis. These isoforms differ both quantitatively and qualitatively. Most remarkably, while the small c-MycS form truncated for much of its N-terminal trans-activation domain efficiently rescued viability and cell growth, it did not induce detectable programmed cell death. Our data indicate that the main functional difference between c-Myc isoforms resides in their apoptotic properties and that the N-terminal region, containing the conserved MbI motif, is decisive in governing the choice between growth and death

Prognostic value of metabolic parameters and clinical impact of 18F-fluorocholine PET/CT in biochemical recurrent prostate cancer

International audiencePURPOSE: To evaluate the therapeutic impact of (18)F-fluorocholine (FCH) PET/CT in biochemical recurrent prostate cancer (PC) and to investigate the value of quantitative FCH PET/CT parameters in predicting progression-free survival (PFS).METHODS: This retrospective study included 172 consecutive patients with PC who underwent FCH PET/CT for biochemical recurrence. Mean rising PSA was 10.7 ± 35.0 ng/ml. Patients with positive FCH PET were classified into three groups: those with uptake only in the prostatic bed, those with locoregional disease, and those with distant metastases. Referring physicians were asked to indicate the hypothetical therapeutic strategy with and without the FCH PET/CT results. Clinical variables and PET parameters including SUVmax, SUVpeak, SUVmean, total lesion choline kinase activity (TLCKA) and standardized added metabolic activity (SAM) were recorded and a multivariate analysis was performed to determine the factors independently predicting PFS.RESULTS: In 137 of the 172 patients, the FCH PET/CT scan was positive, and of these, 29.9 % (41/137) had prostatic recurrence, 42.3 % (58/137) had pelvic lymph node recurrence with or without prostatic recurrence, and 27.7 % (38/137) had distant metastases. The FCH PET/CT result led to a change in treatment plan in 43.6 % (75/172) of the 172 patients. Treatment was changed in 49.6 % (68/137) of those with a positive FCH PET/CT scan and in 20 % (7/35) of those with a negative FCH PET/CT scan. After a median follow-up of 29.3 months (95 % CI 18.9 - 45.9 months), according to multivariate analysis age <70 years, SAM ≥23 and SUVmean ≥3 were parameters independently predicting PFS. A nomogram constructed using the three parameters showed 49 months of PFS in patients with the best scores (0 or 1) and only 11 months in patients with a poor score (score 3).CONCLUSION: This study indicates that a positive FCH PET result in PC patients with biochemical recurrence predicts a shorter PFS and confirms the major impact of the FCH PET result on the management of biochemical recurrent PC