Maurizio Gatti

In animal systems, the only case of third meiotic division without a prior round of DNA replication has been described in Drosophila males carrying mutations in roughex, a gene encoding an inhibitor of Cdk1 activity. Here, we describe another Drosophila gene, terno (teo), that regulates meiotic exit in males. In teo mutants, meiosis I and II are regular, but in many germline cysts, the 64 haploid spermatids undergo an extra division. Additionally, teo mutants are defective in sperm individualization. Teo interacts with cyclin A and B, Fizzy (Cdc20), and the anaphase-promoting complex/cyclosome components Cdc16 and Cdc27. In teo mutant testes, the cyclin A and B levels are higher than in wild type, and downregulation of either cyclin rescues the teo mutant phenotypes. These results suggest that Teo facilitates targeted degradation of the cyclins, so that in teo mutants, there is an increase in Cdk1-CycA and Cdk1-CycB activities, resulting in both the extra meiotic division and the sperm maturation defect.

In animal cells, proper spindle assembly requires generation of large numbers of microtubules (MTs) through three main pathways: MT nucleation by the centrosomes, nucleation near chromosomes/kinetochores and augmin-mediated nucleation from the walls of preexisting MTs. Here, we review the roles of these pathways in spindle assembly in different Drosophila tissues and cell types, with a focus on the chromosome/kinetochore-driven pathway. In female meiosis, there are no centrosomes, and the origin of spindle MTs is currently unclear. In embryos, larval brains and S2 cultured cells, the MTs generated by the mitotic chromatin are sufficient for the assembly of functional spindles, with different contributions of the augmin-dependent MTs. In contrast, in male meiotic cells the chromatin-driven MTs cannot form a functional spindle in the absence of centrosomal MTs, which can assemble a spindle even in the absence of chromosomes. Interestingly, in S2 cells chromatin-driven MTs are generated at kinetochores, as occurs in mammalian cells, while in embryos and larval brains they grow around chromosomes; in male meiotic cells MTs are generated both at kinetochores and around chromosomes. These results indicate that in Drosophila there is a remarkable flexibility in both the modes of chromatin-induced MT nucleation and the usage of MT generation pathways in spindle assembly. It remains to be determined whether a similar flexibility exists in other organisms, and to understand the molecular mechanisms that restrict MT nucleation at kinetochores in S2 cells. These studies will likely contribute to the elucidation of the mechanisms underlying kinetochore-induced MT formation in human cells.

The CMG (Cdc45-MCM-GINS) complex is a conserved helicase that plays an essential DNA unwinding function at replication forks. Here, we analyzed the mitotic phenotypes caused in Drosophila by knockdown of Cdc45, Mcm5, and the four GINS genes (Sld5, Psf1, Psf2, and Psf3). Silencing of these genes resulted in virtually identical mitotic phenotypes. Brain cells from mutant and RNAi larvae showed severe defects in chromosome condensation, chromosome breakage, and frequent polyploid mitotic figures. In addition, mutant cells showed reduced Cid (Cenp-A) incorporation at centromeres and strong alterations in spindle and centrosome structures. Our cytological and genetic analyses suggest that replication-related DNA damage and Cid-dependent centromere/kinetochore defects trigger the spindle assembly checkpoint (SAC) that arrests the cells in a prometaphase-like stage. The arrested cells undergo mitotic slippage accompanied by Cyclin B degradation and eventually return to G1 giving rise to polyploid cells. Our analyses further suggest that during the prolonged prometaphase arrest both the centrosomes and the spindles undergo severe structural degeneration and that the spindle defects are not the consequence of the aberrant centrosome behavior. Most studies on mitotic slippage have been carried out in cells exposed to anti-microtubule agents and could not address the behavior of the spindle. Conversely, our results illuminate the complex consequences of replication stress and reveal what happens to the mitotic apparatus during the prolonged spindle assembly checkpoint–induced mitotic arrest. Because prolonged mitosis is a common event in human cancers, our results could provide useful information for studies on cancer etiology and therapy.

This article provides an overview of Quebec's French-speaking native literature, which is currently becoming more widespread. Its birth and the obstacles encountered over the years are analyzed, with a late affirmation, after the 1970s, due to the historically conflicting relationship between the indigenous peoples and the Quebec school system, and facilitated by the relative sedentarization provided by the creation of the first reservations. We demonstrate how its original manifestations, of a more political and essayistic nature, evolved to encompass several other literary genres, asserting itself today as a space for research and aesthetic creation. We identifie the main themes addressed by the writers, their characteristics and style. We addresse the difficulties of publishing and disseminating the works, and various limitations that lead authors to write mainly in French and not in their native languages. Finally, the achievements of native writers in this historical path are highlighted, as well as the legitimation strategies they adopted and the literary infrastructure they built. O presente artigo traça um panorama geral da literatura nativa francófona do Quebec, que atualmente tem se tornado mais difundida. Analisa-se seu nascimento e os obstáculos por ela encontrados ao longo dos anos, com uma afirmação tardia, após os anos 1970, em razão da relação historicamente conflituosa dos povos autóctones com o sistema escolar quebequense, e facilitada pela relativa sedentarização propiciada pela criação das primeiras reservas. Demonstra-se como suas manifestações originais, de cunho mais político e ensaístico, evoluíram para abarcar diversos outros gêneros literários, afirmando-se hoje como espaço de pesquisa e criação estética. Identifica-se os principais temas abordados pelos escritores, suas características e estilo. Aborda-se as dificuldades de publicação e difusão das obras, e diversas limitações que levam os autores a escrever sobretudo em língua francesa e não em suas línguas nativas. Por fim, sublinha-se as conquistas dos escritores nativos nesse percurso histórico, as estratégias de legitimação por eles adotadas e a infraestrutura literária que construíram.

The Drosophila abnormal spindle (asp) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM (Abnormal Spindle-like Microcephaly-associated; or MCPH5) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse (Aspm) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp/Aspm/ASPM genes play the same conserved functions in Drosophila, mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM), and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast / orbit / chb ( CLASP1 ), mei-38 ( TPX2 ), mars ( HURP ), dgt6 ( HAUS6 ), Eb1 ( MAPRE1/EB1 ), Patronin ( CAMSAP2 ), asp ( ASPM ) and Klp10A ( KIF2A ). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed. Author summary: The mitotic spindle is a microtubule (MT)-based molecular machine that mediates precise chromosome segregation during cell division. Both Drosophila and human cells assemble their spindles exploiting two main classes of MTs: MTs nucleated by the centrosomes (MT nucleating organelles) and MTs generated at or near the kinetochores (the chromosome-associated structures that bind the spindle MTs). Cells of both species can assemble a functional mitotic spindle in the complete absence of centrosomes, but the mechanisms underlying this process are still poorly understood. We used Drosophila S2 cells as model system to analyze spindle reassembly following colcemid-induced MT depolymerization. MT regrowth (MTR) after colcemid treatment was particularly informative as this drug disrupts the MT nucleating ability of the centrosomes but allows kinetochore-driven MTR (KDMTR). We analyzed KDMTR in normal cells and in cells subjected to RNA interference (RNAi)-mediated depletion of 8 different evolutionarily conserved proteins involved in spindle assembly, and identified proteins that either promote or delay KDMTR. These results coupled with the analysis of proteins localization during spindle reassembly allowed us to integrate the current model on the role of kinetochore-driven MT growth in spindle formation.

Moonlighting proteins can perform one or more additional functions besides their primary role. It has been posited that a protein can acquire a moonlighting function through a gradual evolutionary process, which is favored when the primary and secondary functions are exerted in different cellular compartments. Transcription factors (TFs) and splicing factors (SFs) control processes that occur in interphase nuclei and are strongly reduced during cell division, and are therefore in a favorable situation to evolve moonlighting mitotic functions. However, recently published moonlighting protein databases, which comprise almost 400 proteins, do not include TFs and SFs with secondary mitotic functions. We searched the literature and found several TFs and SFs with bona fide moonlighting mitotic functions, namely they localize to specific mitotic structure(s), interact with proteins enriched in the same structure(s), and are required for proper morphology and functioning of the structure(s). In addition, we describe TFs and SFs that localize to mitotic structures but cannot be classified as moonlighting proteins due to insufficient data on their biochemical interactions and mitotic roles. Nevertheless, we hypothesize that most TFs and SFs with specific mitotic localizations have either minor or redundant moonlighting functions, or are evolving towards the acquisition of these functions.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease. We explored the functional relationships between TGS1 and SMN using Drosophila as model organism. TGS1 is an enzyme that modifies the structure of the 5’-end of several RNAs, including telomerase RNA and the small nuclear RNAs (snRNAs) that are required for messenger RNA maturation. The SMN protein regulates snRNAs biogenesis and mutations in human SMN cause Spinal Muscular Atrophy (SMA), a devastating disorder characterized by neurodegeneration, progressive paralysis and death. We show that mutations in the Drosophila TGS1 (dTgs1) gene cause lethality, which is rescued by a human TGS1 transgene. We also show that the dTgs1 protein physically interacts with all subunits of the Smn complex, and that downregulation of either dTgs1 or Smn leads to a reduced Drosophila eye size. Notably, overexpression of dTgs1 partially rescues the eye defects caused by Smn knockdown, and vice versa, indicating that these genes cooperate in eye development. These results suggest that the eye model can be exploited for screens aimed at detection of chemical and genetic modifiers of the eye mutant phenotype elicited by dTgs1 and Smn deficiency, providing new clues about SMA pathogenesis and potential therapies.