Mob4 is required for neurodevelopment in zebrafish.

Mob4 is an essential evolutionary conserved protein shown to play roles in cell division and neural development. Mob4 is a core component of the macromolecular STRIPAK complex involved in various critical cellular processes, from cell division to signal transduction pathways. However, Mob4 remains relatively poorly understood. Although the consequences of eliminating Mob4 function in Drosophila are described, its function in vertebrate development remains largely unknown. Here we show that knockdown and knockout of Mob4 during zebrafish embryogenesis limits neuronal cell divisions but has little effect on apoptosis, thus arguing a role for mob4 in neurodevelopment.

Spindly and Bub3 expression in oral cancer: Prognostic and therapeutic implications.

OBJECTIVES: Bub3 and Spindly are essential proteins required for the activation and inactivation of the spindle assembly checkpoint, respectively. Here, we explored the clinicopathological significance and the therapeutic potential of the opposing roles of the two proteins in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Bub3 and Spindly expression was evaluated by immunohistochemistry in 62 tissue microarrays from OSCC and by real-time PCR in OSCC cell lines and in normal human oral keratinocytes. The results were analyzed as to their clinicopathological significance. RNA interference-mediated Spindly or Bub3 inhibition was combined with cisplatin treatment, and the effect on the viability of OSCC cells was assessed. RESULTS: Overexpression of Bub3 and Spindly was detected in OSCC patients. High expression of Spindly, Bub3, or both was an independent prognostic indicator for cancer-specific survival and was associated with increased cellular proliferation. Accordingly, Bub3 and Spindly were upregulated in OSCC cells comparatively to their normal counterpart. Inhibition of Bub3 or Spindly was cytotoxic to OSCC cells and enhanced their chemosensitivity to cisplatin. CONCLUSIONS: The data point out Bub3 and Spindly as potential markers of proliferation and prognosis, and highlight the potential therapeutic benefit of combining their inhibition with cisplatin.

Report of in vitro antileishmanial properties of Iberian macroalgae.

Here is reported the anti Leishmania infantum activity of 48 hexane, CH2Cl2 and MeOH extracts from 16 macroalgae collected on the Iberian Coast. Seven hexane and CH2Cl2 Cystoseira baccata, Cystoseira barbata, Cystoseira tamariscifolia, Cystoseira usneoides, Dictyota spiralis and Plocamium cartilagineum extracts were active towards promastigotes (IC50 29.8-101.8 µg/mL) inducing strong morphological alterations in the parasites. Hexane extracts of C. baccata and C. barbata were also active against intracellular amastigotes (IC50 5.1 and 6.8 µg/mL, respectively). Fatty acids, triacylglycerols, carotenoids, steroids and meroterpenoids were detected by nuclear magnetic resonance (NMR), and gas chromatography in the Cystoseira extracts. These results suggest that Cystoseira macroalgae contain compounds with antileishmanial activity, which could be explored as scaffolds to the development of novel sources of antiparasitic derivatives.

A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosis.

Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.

Calpastatin Overexpression Preserves Cognitive Function Following Seizures, While Maintaining Post-Injury Neurogenesis.

In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled.

Suppression of spindly delays mitotic exit and exacerbates cell death response of cancer cells treated with low doses of paclitaxel.

Microtubule-targeting agents (MTAs) are used extensively for the treatment of diverse types of cancer. They block cancer cells in mitosis through the activation of the spindle assembly checkpoint (SAC), the surveillance mechanism that ensures accurate chromosome segregation at the onset of anaphase. However, the cytotoxic activity of MTAs is limited by premature mitotic exit (mitotic slippage) due to SAC silencing. Here we have explored the dual role of the protein Spindly in chromosome attachments and SAC silencing to analyze the consequences of its depletion on the viability of tumor cells treated with clinically relevant doses of paclitaxel. As expected, siRNA-mediated Spindly suppression induced chromosome misalignment and accumulation of cells in mitosis. Remarkably, these cells were more sensitive to low-doses of paclitaxel. Sensitization was due to an increase in the length of mitotic arrest and high frequency of multinucleated cells, both correlated with an exacerbated post-mitotic cell death response as determined by cell fate profiling. Thus, by affecting both SAC silencing and chromosome attachment, Spindly targeting offers a double-edged sword that potentiates tumor cell killing by clinically relevant doses of paclitaxel, providing a rationale for combination chemotherapy against cancer.

Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles.

A predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediated transport of certain kinetochore proteins along microtubules. There are still conflicting data as to which SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the core SAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spindle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arrested cells, at a time when the SAC should be satisfied and silenced. Unexpectedly, we found that a pool of Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additional dynein cargoes. The potential significance of these results for SAC silencing is discussed.

Maternal thyroid hormones are essential for neural development in zebrafish.

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

Human Mob1 proteins are required for cytokinesis by controlling microtubule stability.

The completion of cytokinesis requires abscission of the midbody, a microtubule-rich cytoplasmic bridge that connects the daughter cells before their final separation. Although it has been established that both the midbody structure and membrane fusion are essential for abscission, the biochemical machinery and the cellular processes of abscission remain ill-defined. Here we report that human Mob1A and Mob1B proteins are involved in the regulation of abscission of the intercellular bridge. The Mob family is a group of highly conserved proteins in eukaryotes, described as binding partners as well as co-activators of protein kinases of the Ndr family, and as members of the Hippo pathway. We show that depletion of Mob1A and Mob1B by RNAi causes abscission failure as a consequence of hyper-stabilization of microtubules in the midbody region. Interestingly, depleting Mob1 also increases cell motility after cytokinesis, and induces prolonged centriole separation in G1 phase. In contrast, centrosomes fail to split when either Mob1A or Mob1B is overexpressed. Our findings indicate that human Mob1 proteins are involved in the regulation of microtubule stability at the midbody. We conclude that Mob1A and Mob1B are needed for cell abscission and centriole re-joining after telophase and cytokinesis.

Mob1: defining cell polarity for proper cell division.

Mob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior-posterior axis, left-right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role of Tetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.

Overexpression of four and a half LIM domains protein 2 promotes epithelial-mesenchymal transition-like phenotype in fish pre-osteoblasts.

FHL2 is a multifunctional protein involved in gene transcription regulation and cytoarchitecture modulation that has been recently associated with epithelial-mesenchymal transition (EMT) in colon cancer. Overexpression of FHL2 in a fish pre-osteoblastic cell line promoted cell dedifferentiation and impaired its extracellular matrix mineralization capacity. Cell cultures also acquired a novel three-dimensional structure organization, their proliferation rate was enhanced and gene expression profile was altered in agreement with an EMT-like phenotype upon overexpression of FHL2. Altogether, our results provide additional support to the relevance of FHL2 for cell differentiation and its association with hallmarks of cancer phenotype.

Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae.

We describe a new selection method based on BODIPY (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about 3 weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla.