Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.     

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer <Emphasis Type="Italic">Streptomyces collinus</Emphasis> Tü 365

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.     

Live-cell fluorescence imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the entirety of microgametogenesis. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach captured early microgametogenesis with three-dimensional imaging through time (4D imaging) and microgamete release with two-dimensional (2D) fluorescence microscopy. To minimise the phototoxic impact to parasites, acquisition was alternated between 4D fluorescence, brightfield and 2D fluorescence microscopy. Combining live-cell dyes specific for DNA, tubulin and the host erythrocyte membrane, 4D and 2D imaging together enables definition of the positioning of newly replicated and segregated DNA. This combined approach also shows the microtubular cytoskeleton, location of newly formed basal bodies, elongation of axonemes and morphological changes to the erythrocyte membrane, the latter including potential echinocytosis of the erythrocyte membrane prior to microgamete egress. Extending the utility of this approach, the phenotypic effects of known transmission-blocking inhibitors on microgametogenesis were confirmed. Additionally, the effects of bortezomib, an untested proteasomal inhibitor, revealed a clear block of DNA replication, full axoneme nucleation and elongation. Thus, as well as defining a framework for broadly investigating microgametogenesis, these data demonstrate the utility of using live imaging to validate potential targets for transmission-blocking antimalarial drug development.     

Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy

Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.     

Potential clinical implications of recent matrix metalloproteinase inhibitor design strategies

Analysis of matrix metalloproteinases (MMPs) expression profiles in various pathologies correlated with their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site zinc ion. This approach did not distinguish between the 24 members of the MMP family and had devastating consequences during clinical trials. Subsequent knockout mouse studies showed that some MMPs are beneficial in regulating tumor growth, metastasis and indirectly stimulating the immune system. The broad-spectrum inhibitor approach was rethought and modified in order to increase specificity by taking into account the non-conserved secondary binding sites or differences in structures within MMPs and also generating antibodies. These showed interesting results in vitro and in vivo. The recent technological advances that allow us to better understand the function and structure of MMPs are aiding in the development of selective inhibitors.     

The Polo-Like Kinase 1 (PLK1) Inhibitor NMS-P937 Is Effective in a New Model of Disseminated Primary CD56+ Acute Monoblastic Leukaemia

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56⁺ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56⁺ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.     

BCR-ABL Promotes PTEN Downregulation in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor PTEN plays a critical role in the pathogenesis of CML chronic phase, through non genomic loss of function mechanisms, such as protein down-regulation and impaired nuclear/cytoplasmic shuttling. Here we demonstrate that BCR-ABL promotes PTEN downregulation through a MEK dependent pathway. Furthermore, we describe a novel not recurrent N212D-PTEN point mutation found in the EM2 blast crisis cell line.