ABSTRACT
Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.
Subject(s)
Gene Editing/methods , Sex Determination Processes/genetics , Sex Preselection/methods , Animals , CRISPR-Cas Systems/genetics , DNA End-Joining Repair/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Endonucleases/genetics , Female , Male , Models, Animal , Pest Control, Biological/methods , Sex Ratio , Spermatogenesis/genetics , X Chromosome/geneticsABSTRACT
BACKGROUND: Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. RESULTS: Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%). CONCLUSION: We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.
Subject(s)
Ceratitis capitata , Animals , Animals, Genetically Modified , CRISPR-Cas Systems/genetics , Ceratitis capitata/genetics , Female , Male , RNA, Guide, Kinetoplastida , Sex Ratio , X Chromosome/geneticsABSTRACT
dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.
Subject(s)
Adenosine Triphosphatases/genetics , Autoantigens/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Nucleosomes/genetics , Polytene Chromosomes/genetics , Adenosine Triphosphatases/metabolism , Animals , Autoantigens/metabolism , Cell Cycle Proteins/metabolism , Chromatids , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Fluorescence Recovery After Photobleaching , Interphase/genetics , Protein Binding , Salivary Glands/cytology , Salivary Glands/metabolism , CohesinsABSTRACT
RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression-based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression-based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.
Subject(s)
Drosophila/genetics , Gene Expression , Genes, Insect , Mitosis/genetics , RNA Interference , Animals , Chromosome Segregation , Cytokinesis , Drosophila/metabolism , RNA, Double-Stranded/metabolism , Spindle Apparatus/genetics , Spindle Apparatus/metabolismABSTRACT
Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows.
Subject(s)
Cell Cycle , Drosophila Proteins/metabolism , Dyneins/metabolism , Microtubule-Organizing Center/metabolism , Nuclear Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Anaphase , Animals , Centrosome/metabolism , Drosophila melanogaster/embryology , Embryo, Nonmammalian/cytology , Immunoprecipitation , Microtubules/metabolism , Prophase , Protein Binding , Protein Transport , TelophaseABSTRACT
A longstanding enigma has been the origin of maternal centrosomes that facilitate parthenogenetic development in Hymenopteran insects. In young embryos, hundreds of microtubule-organizing centers (MTOCs) are assembled completely from maternal components. Two of these MTOCs join the female pronucleus to set up the first mitotic spindle in unfertilized embryos and drive their development. These MTOCs appear to be canonical centrosomes because they contain gamma-tubulin, CP190, and centrioles and they undergo duplication. Here, we present evidence that these centrosomes originate from accessory nuclei (AN), organelles derived from the oocyte nuclear envelope. In the parasitic wasps Nasonia vitripennis and Muscidifurax uniraptor, the position and number of AN in mature oocytes correspond to the position and number of maternal centrosomes in early embryos. These AN also contain high concentrations of gamma-tubulin. In the honeybee, Apis mellifera, distinct gamma-tubulin foci are present in each AN. Additionally, the Hymenopteran homolog of the Drosophila centrosomal protein Dgrip84 localizes on the outer surfaces of AN. These organelles disintegrate in the late oocyte, leaving behind small gamma-tubulin foci, which likely seed the formation of maternal centrosomes. Accessory nuclei, therefore, may have played a significant role in the evolution of haplodiploidy in Hymenopteran insects.
Subject(s)
Centrosome , Hymenoptera/physiology , Parthenogenesis/physiology , Animals , Drosophila Proteins/immunology , Female , Microtubule-Associated Proteins/immunology , Nuclear Envelope/physiology , Oocytes/ultrastructure , Oogenesis/physiology , Organelles/metabolism , Tubulin/metabolismABSTRACT
ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.
Subject(s)
Body Patterning/physiology , Chromosomal Instability/physiology , DNA Repair , Drosophila/growth & development , Protein Serine-Threonine Kinases/metabolism , Telomere/physiology , Animals , Apoptosis/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins , Crosses, Genetic , DNA-Binding Proteins , Drosophila/ultrastructure , Eye/pathology , Larva/growth & development , Larva/ultrastructure , Locomotion/physiology , Microscopy, Electron , Mutagenesis , Mutation/genetics , Neurons/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Recombination, Genetic/physiology , Temperature , Transgenes/genetics , Tumor Suppressor Proteins , Wings, Animal/pathologyABSTRACT
We have used double-stranded RNA-mediated interference (RNAi) to study Drosophila cytokinesis. We show that double-stranded RNAs for anillin, acGAP, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. Our phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and lead us to propose that the central spindle and the contractile ring are interdependent structures. Finally, our results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis.
Subject(s)
Cell Division/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/physiology , Genes, cdc , RNA Interference , Actins/metabolism , Animals , Cell Division/physiology , Cell Membrane/metabolism , Cell Separation , Cells, Cultured , Contractile Proteins/metabolism , Cytoskeletal Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Flow Cytometry , Immunohistochemistry , Microtubules/metabolism , Phenotype , Spindle Apparatus/metabolismABSTRACT
Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. Here we demonstrate that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes.
Subject(s)
Drosophila Proteins/metabolism , GTPase-Activating Proteins/metabolism , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Molecular Motor Proteins/metabolism , Adenosine Triphosphatases/metabolism , Animals , Blotting, Western , Cell Line , Centrosome/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , GTPase-Activating Proteins/genetics , Kinesins/genetics , Kinetics , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Molecular Motor Proteins/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , RNA InterferenceABSTRACT
Barren, the Drosophila homolog of XCAP-H, is one of three non-SMC subunits of condensin, a conserved 13S multiprotein complex required for chromosome condensation. Mutations in barren (barr) were originally shown to affect sister-chromatid separation during mitosis 16 of the Drosophila embryo, whereas condensation defects were not detected. In contrast, mutations in yeast homologs of barren result in defective mitotic chromosome condensation as well as irregular chromatid separation. We have used double-stranded RNA-mediated interference (RNAi) to deplete Barren in Drosophila S2 cells. Our analyses indicate that inactivation of barr leads to extensive chromosome condensation and disrupts chromatid segregation.
Subject(s)
Cell Cycle Proteins/genetics , Drosophila Proteins/genetics , Drosophila/genetics , RNA Interference , Animals , Base Sequence , DNA Primers , In Situ Hybridization, Fluorescence , MutationABSTRACT
The model organism Drosophila melanogaster is particularly well suited for live image analysis. The availability of GFP transgenic flies and a wide array of fluorescent probes, in conjunction with laser scanning confocal microscopy, allow us to image multiple aspects of the cell cycle simultaneously. Confocal microscopy provides the sensitivity and resolution to observe the dynamics of specific cellular events in real time. For example, GFP-histone and rhodamine-labeled tubulin enable one to follow specific nuclear and cytoskeletal events including nuclear envelope formation, nuclear envelope breakdown, spindle formation, centrosome duplication, separation and migration, chromosomes condensation, and segregation. This analysis permits a detailed morphological and temporal description of nuclear and cytoskeletal events in normal or drug-injected embryos.
Subject(s)
Drosophila/embryology , Embryo, Nonmammalian/ultrastructure , Green Fluorescent Proteins , Rhodamines , Animals , Animals, Genetically Modified , Drosophila/drug effects , Embryo, Nonmammalian/drug effects , Mitosis/drug effects , Mutation , Nuclear Envelope/drug effects , Nuclear Envelope/ultrastructure , Staining and Labeling , Tubulin/metabolismABSTRACT
Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events.
Subject(s)
Anaphase , Cell Cycle Proteins/metabolism , Chromosomes/metabolism , DNA Replication , Nuclear Proteins/metabolism , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Checkpoint Kinase 1 , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Nuclear Envelope/metabolism , S Phase , Topoisomerase Inhibitors/pharmacologyABSTRACT
Proper centrosome separation is a prerequisite for positioning the bipolar spindle. Although studies demonstrate that microtubules (MTs) and their associated motors drive centrosome separation [1], the role of actin in centrosome separation remains less clear. Studies in tissue culture cells indicate that actin- and myosin-based cortical flow is primarily responsible for driving late centrosome separation [2], whereas other studies suggest that actin plays a more passive role by serving as an attachment site for astral MTs to pull centrosomes apart [3-6]. Here we demonstrate that prior to nuclear envelope breakdown (NEB) in Drosophila embryos, proper centrosome separation does not require myosin II but requires dynamic actin rearrangements at the growing edge of the interphase cap. Both Arp2/3- and Formin-mediated actin remodeling are required for separating the centrosome pairs before NEB. The Apc2-Armadillo complex appears to link cap expansion to centrosome separation. In contrast, the mechanisms driving centrosome separation after NEB are independent of the actin cytoskeleton and compensate for earlier separation defects. Our studies show that the dynamics of actin polymerization drive centrosome separation, and this has important implications for centrosome positioning during processes such as cell migration [7, 8], cell polarity maintenance [9, 10], and asymmetric cell division [11, 12].
Subject(s)
Actins/metabolism , Cell Cycle/physiology , Centrosome/metabolism , Cytoskeleton/metabolism , Spindle Apparatus/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Actins/ultrastructure , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Formins , Microtubules/metabolism , Myosin Type II/genetics , Myosin Type II/metabolism , Nuclear Envelope/metabolism , Tubulin/genetics , Tubulin/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. Here we show that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. Our results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition.