Eph signaling in mitotic spindle orientation: what´s your angle here?

The orientation of the mitotic spindle is a crucial process during development and adult tissue homeostasis and multiple mechanisms have been shown to intrinsically regulate this process. However, much less is known about the extrinsic cues involved in modulating spindle orientation. We have recently uncovered a novel function of Eph intercellular signaling in regulating spindle alignment by ultimately ensuring the correct cortical distribution of central components within the intrinsic spindle orientation machinery. Here, we comment on these results, novel questions that they open and potential additional research to address in the future.

Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells.

Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. Here, we uncover a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. We show that conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.

Measurement of Mitotic Spindle Angle and Mitotic Cell Distance in Fixed Tissue of Drosophila Larval Brains.

The positioning and the cleavage plane orientation of mitotic cells in pseudostratified epithelia (PSE) must be tightly regulated since failures in any of these processes might have fatal consequences during development. Here we present a simple method to determine the spindle orientation as well as the positioning of neuroepithelial mitotic cells within the Outer Proliferation Center (OPC) of Drosophila larval brains.

Synergism between canoe and scribble mutations causes tumor-like overgrowth via Ras activation in neural stem cells and epithelia.

Over the past decade an intriguing connection between asymmetric cell division, stem cells and tumorigenesis has emerged. Neuroblasts, which are the neural stem cells of the Drosophila central nervous system, divide asymmetrically and constitute an excellent paradigm for investigating this connection further. Here we show that the simultaneous loss of the asymmetric cell division regulators Canoe (afadin in mammals) and Scribble in neuroblast clones leads to tumor-like overgrowth through both a severe disruption of the asymmetric cell division process and canoe loss-mediated Ras-PI3K-Akt activation. Moreover, canoe loss also interacts synergistically with scribble loss to promote overgrowth in epithelial tissues, here just by activating the Ras-Raf-MAPK pathway. discs large 1 and lethal (2) giant larvae, which are functionally related to scribble, contribute to repress the Ras-MAPK signaling cascade in epithelia. Hence, our work uncovers novel cooperative interactions between all these well-conserved tumor suppressors that ensure tight regulation of the Ras signaling pathway.

The hippo pathway core cassette regulates asymmetric cell division.

Asymmetric cell division (ACD) is a crucial process during development, homeostasis, and cancer. Stem and progenitor cells divide asymmetrically, giving rise to two daughter cells, one of which retains the parent cell self-renewal capacity, while the other is committed to differentiation. Any imbalance in this process can induce overgrowth or even a cancer-like state. Here, we show that core components of the Hippo signaling pathway, an evolutionarily conserved organ growth regulator, modulate ACD in Drosophila. Hippo pathway inactivation disrupts the asymmetric localization of ACD regulators, leading to aberrant mitotic spindle orientation and defects in the generation of unequal-sized daughter cells. The Hippo pathway downstream kinase Warts, LATS1-2 in mammals, associates with the ACD modulators Inscuteable and Bazooka in vivo and phosphorylates Canoe, the ortholog of Afadin/AF-6, in vitro. Moreover, phosphosite mutant Canoe protein fails to form apical crescents in dividing neuroblasts in vivo, and the lack of Canoe phosphorylation by Warts leads to failures of Discs Large apical localization in metaphase neuroblasts. Given the relevance of ACD in stem cells during tissue homeostasis, and the well-documented role of the Hippo pathway as a tumor suppressor, these results represent a potential route for perturbations in the Hippo signaling to induce tumorigenesis via aberrant stem cell divisions.

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.

BACKGROUND: Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. CONCLUSIONS/SIGNIFICANCE: These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10.

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.

A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.

A third functional isoform enriched in mushroom body neurons is encoded by the Drosophila 14-3-3zeta gene.

14-3-3 proteins are highly conserved across eukaryotes, typically encoded by multiple genes in most species. Drosophila has only two such genes, 14-3-3zeta (leo), encoding two isoforms LEOI and LEOII, and 14-3-3epsilon. We report a bona fide third functional isoform encoded by leo divergent from the other two in structurally and functionally significant areas, thus increasing 14-3-3 diversity in Drosophila. Furthermore, we used a novel approach of spatially restricted leo abrogation by RNA-interference and revealed differential LEO distribution in adult heads, with LEOIII enrichment in neurons essential for learning and memory in Drosophila.

[Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)]. / Distribución y sensibilidad a los antifúngicos de aislamientos clínicos de Candida en seis centros de salud del área metropolitana de Caracas, Venezuela (años 2003-2005).

The aim of this study was to investigate the frequency and antifungal susceptibility of Candida clinical isolations coming from patients with candidiasis in six health care centers of Caracas, Venezuela metropolitan area. The laboratory reports were retrospectively revised from January 2003 through August 2005. The isolated yeasts identification was carried out by conventional methods and antifungal susceptibility was evaluated by ATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden). One thousand nine hundred seventy seven (1.977) yeasts were studied and their susceptibility testing were carried out only in 1,414 of them. C. albicans was the most isolated yeast (46.7%) and none-albicans Candida-species represented more than half of the isolations (53.4%). All the isolated yeasts evaluated presented CMIs<1 microg/ml to anfotericina B and showed variable susceptibility percentages to fluconazole (91.5%), itraconazole (80%) and voriconazole (98.6%).

Increased mitochondrial DNA copy-number in CEM cells resistant to delayed toxicity of 2',3'-dideoxycytidine.

The nucleoside analog 2',3'-dideoxycytidine (ddC) has been used for treatment of human immunodeficiency virus (HIV) infections. ddC causes delayed toxicity when cells are exposed to the drug at low concentration for prolonged periods of time. The delayed toxicity is due to inhibition of mitochondrial DNA (mtDNA) replication, which results in mtDNA depletion and mitochondrial dysfunction. In the present study we have cultured CEM T-lymphoblast cells in the presence of low concentrations of ddC to generate two cell lines resistant to the delayed toxicity of the drug. Both cell lines were resistant to mtDNA depletion by ddC. The mechanism of ddC resistance was investigated and we showed that the resistant cells had decreased mRNA expression of the nucleoside kinases deoxycytidine kinase and thymidine kinase 2. We also studied the mitochondrial DNA in the cells and showed that the ddC-resistant cells had structurally intact mtDNA but 1.5-2-fold increased mtDNA copy-number as well as increased levels of the mitochondrial transcription factor A (Tfam). Our study suggests that cells may increase their level of mtDNA to counteract mtDNA depletion induced by ddC, while keeping pronounced antiviral activity of the drug.

Distribución y sensibilidad a los antifúngicos de aislamientos clínicos de Candida en seis centros de salud del área metropolitana de Caracas, Venezuela (años 2003-2005) / Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)

El objetivo de este estudio fue conocer la frecuencia y la sensibilidad a losantifúngicos de aislamientos clínicos de Candida provenientes de pacientescon candidiasis en seis centros de salud del área metropolitana de Caracas,Venezuela. Se revisaron retrospectivamente los informes de laboratorio desdeenero de 2003 hasta agosto de 2005. La identificación de las levadurasaisladas se realizó por los métodos convencionales y se evaluó lasusceptibilidad a los antifúngicos por los métodos ATB-Fungus (bioMérieux,Francia) y Etest (AB Biodisk, Solna, Suecia). Se aislaron 1.977 levaduras y a1.414 se les realizaron pruebas de sensibilidad. Candida albicans fue lalevadura aislada con más frecuencia (46,7%) y el resto de las especies deCandida representaron más de la mitad de los aislamientos (53,4%).Todas las levaduras evaluadas presentaron valores de CMI <1 mg/ml para laanfotericina B y porcentajes de sensibilidad variable al fluconazol (91,5%),itraconazol (80%) y voriconazol (98,6%)(AU)

The aim of this study was to investigate the frequency and antifungalsusceptibility of Candida clinical isolations coming from patients withcandidiasis in six health care centers of Caracas, Venezuela metropolitan area.The laboratory reports were retrospectively revised from January 2003 throughAugust 2005. The isolated yeasts identification was carried out byconventional methods and antifungal susceptibility was evaluated byATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden).One thousand nine hundred seventy seven (1,977) yeasts were studied andtheir susceptibility testing were carried out only in 1,414 of them. C. albicanswas the most isolated yeast (46.7%) and none-albicans Candida-speciesrepresented more than half of the isolations (53.4%). All the isolated yeastsevaluated presented CMIs <1 mg/ml to anfotericina B and showed variablesusceptibility percentages to fluconazole (91.5%), itraconazole (80%) andvoriconazole (98.6%)(AU)

Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells.

Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster.

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.

Further variability within the genus Crinivirus, as revealed by determination of the complete RNA genome sequence of Cucurbit yellow stunting disorder virus.

The complete nucleotide (nt) sequences of genomic RNAs 1 and 2 of Cucurbit yellow stunting disorder virus (CYSDV) were determined for the Spanish isolate CYSDV-AlLM. RNA1 is 9123 nt long and contains at least five open reading frames (ORFs). Computer-assisted analyses identified papain-like protease, methyltransferase, RNA helicase and RNA-dependent RNA polymerase domains in the first two ORFs of RNA1. This is the first study on the sequences of RNA1 from CYSDV. RNA2 is 7976 nt long and contains the hallmark gene array of the family Closteroviridae, characterized by ORFs encoding a heat shock protein 70 homologue, a 59 kDa protein, the major coat protein and a divergent copy of the coat protein. This genome organization resembles that of Sweet potato chlorotic stunt virus (SPCSV), Cucumber yellows virus (CuYV) and Lettuce infectious yellows virus (LIYV), the other three criniviruses sequenced completely to date. However, several differences were observed. The most striking novel features of CYSDV compared to SPCSV, CuYV and LIYV are a unique gene arrangement in the 3'-terminal region of RNA1, the identification in this region of an ORF potentially encoding a protein which has no homologues in any databases, and the prediction of an unusually long 5' non-coding region in RNA2. Additionally, the CYSDV genome resembles that of SPCSV in having very similar 3' regions in RNAs 1 and 2, although for CYSDV similarity in primary structures did not result in predictions of equivalent secondary structures. Overall, these data reinforce the view that the genus Crinivirus contains considerable genetic variation. Additionally, several subgenomic RNAs (sgRNAs) were detected in CYSDV-infected plants, suggesting that generation of sgRNAs is a strategy used by CYSDV for the expression of internal ORFs.