61 research outputs found
The Drosophila RZZ complex: roles in membrane traffic and cytokinesis
No full textThe Zw10 protein, in the context of the conserved Rod-Zwilch-Zw10 (RZZ) complex, is a kinetochore component required for proper activity of the spindle assembly checkpoint in both Drosophila and mammals. In mammalian and yeast cells, the Zw10 homologues, together with the conserved RINT1/Tip20p and NAG/Sec39p proteins, form a second complex involved in vesicle transport between Golgi and ER. However, it is currently unknown whether Zw10 and the NAG family member Rod are also involved in Drosophila membrane traffic. Here we show that Zw10 is enriched at both the Golgi stacks and the ER of Drosophila spermatocytes. Rod is concentrated at the Golgi but not at the ER, while Zwilch does not accumulate in any membrane compartment. Mutations in zw10 and RNAi against the Drosophila homologue of RINT1 (rint1) cause strong defects in Golgi morphology and reduce the number of Golgi stacks. Mutations in rod also affect Golgi morphology, while zwilch mutants do not exhibit gross Golgi defects. Loss of either Zw10 or Rint1 results in frequent failures of spermatocyte cytokinesis, whereas Rod or Zwilch are not required for this process. Spermatocytes lacking zw10 or rint1 function assemble regular central spindles and acto-myosin rings, but furrow ingression halts prematurely due to defective plasma membrane addition. Collectively, our results suggest that Zw10 and Rint1 cooperate in the ER-Golgi traffic and in plasma membrane formation during spermatocyte cytokinesis. Our findings further suggest that Rod plays a Golgi-related function that is not required for spermatocyte cytokinesis
Genetics dissection of molecular mechanisms invlved in new membrane formation during cyto kinesis df Drosophila melanogaster.
No full textRoles of the Drosophila NudE protein in kinetochore function and centrosome migration
No full textWe examined the distribution of the dynein-associated protein NudE in Drosophila larval brain neuroblasts and spermatocytes, and analyzed the phenotypic consequences of a nudE null mutation. NudE can associate with kinetochores, spindles and the nuclear envelope. In nudE mutant brain mitotic cells, centrosomes are often detached from the poles. Moreover, the centrosomes of mutant primary spermatocytes do not migrate from the cell cortex to the nuclear envelope, establishing a new role for NudE. In mutant neuroblasts, chromosomes fail to congress to a tight metaphase plate, and cell division arrests because of spindle assembly checkpoint (SAC) activation. The targeting of NudE to mitotic kinetochores requires the dyneininteracting protein Lis1, and surprisingly Cenp-meta, a Drosophila CENP-E homolog. NudE is non-essential for the targeting of all mitotic kinetochore components tested. However, in the absence of NudE, the ‘shedding’ of proteins off the kinetochore is abrogated and the SAC cannot be turned off, implying that NudE regulates dynein function at the kinetochore
Expansion microscopy on Drosophila spermatocyte centrioles
No full textDrosophila spermatocyte centrioles are ideal for imaging studies. Their large, characteristic V conformation is both easy to identify and measure using standard imaging techniques. However, certain detailed features, such as their ninefold symmetry, are only visible below the diffraction limit of light. This is therefore a system that can benefit from the increased effective resolution potentially achievable by expansion microscopy. Here, I provide detailed protocols of two types of expansion microscopy methodologies applied to Drosophila spermatocyte centrioles, and discuss which is able to achieve the highest effective resolution in this system. I describe how to precisely measure these organelles post-expansion, and discuss how they can therefore be used as “molecular rulers” to troubleshoot and compare expansion techniques. I also provide protocols to combine expansion microscopy with super-resolution imaging in this tissue, discussing potential pitfalls. I conclude that expansion microscopy provides an effective alternative for thick tissues that are not amenable for traditional super-resolution techniques
Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster
No full textCytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. We demonstrate that the gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster We show that Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. We further demonstrate that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. We propose that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis
SAPs as a new model to probe the pathway of centriole and centrosome assembly
No full textCentrioles are important cellular organelles involved in the formation of both cilia and centrosomes. It is therefore not surprising that their dysfunction may lead to a variety of human pathologies. Studies have identified a conserved pathway of proteins required for centriole formation, and investigations using the embryo of the fruit fly Drosophila melanogaster have been crucial in elucidating their dynamics. However, a full understanding of how these components interact has been hampered by the total absence of centrioles in null mutant backgrounds for any of these core centriole factors. Here, I review our recent work describing a new model for investigating these interactions in the absence of bona fide centrioles. Sas-6 Ana2 Particles (SAPs) form when two core centriole factors, Sas-6 and Ana2, are co-over-expressed in fruit fly eggs. Crucially, they form even in eggs lacking other core centriole proteins. I review our characterisation of SAPs, and provide one example of how they have been used to investigate the role of a core centriole protein in PCM formation. I then consider some of the strengths and weaknesses of the SAP model, and discuss them in the context of other models for centriole study in Drosophila. Similar aggregates have been seen in other systems upon expression of centriole factors, so SAPs may also be a useful approach to study centriole proteins in other organisms
Quantitative analysis of incorporation dynamics of conserved Centriole proteins in Drosophila early embryos
No full textTo divide efficiently, cells of animals and many other higher eukaryotes, rely upon centrosomes to help nucleate the bipolar mitotic spindle. At each division, two centrosomes are required, such that one centrosome is ultimately inherited by each daughter cell. Consequently, centrosomes must duplicate precisely once during each cycle of cell division. At the core of each centrosome is a mother centriole that can duplicate by growing a new daughter centriole off its side, which will later mature into a mother and nucleate its own centrosome. The core components of the centriole are highly conserved across higher eukaryotes, despite this there is still a lack of detail regarding their function and their hierarchy of assembly.
In this thesis, I perform a comparative analysis of the incorporation dynamics of several conserved centriole proteins, using both standard and super-resolution microscopy, and the Drosophila early embryo as a model. I show that the daughter centrioles in the Drosophila early embryo appear to begin assembling earlier than previously reported. Further, I provide evidence that several core centriole proteins may be recruited into a transient centrosomal primordial “soup” in addition to the centriole proper. In addition, I refine the order of assembly of the centriole - allowing incorporation of Sas-6, Ana2, Ana3, CEP135, Ana1 and Asl to be placed on a continuous timeline alongside the cell cycle. Finally, through the use of CEP135 mutants, I show that, CEP135 does not appear to be necessary to recruit Ana1 into centrioles, in contrast to previous findings.
In summary, my work redefines the initial incorporation dynamics of centriole assembly, challenging several previous assumptions and providing new avenues for interrogation of the specific functions of the core centriole proteins
Mob4 is essential for spermatogenesis in Drosophila melanogaster
No full textGamete formation is essential for sexual reproduction in metazoans. Meiosis in males gives rise to spermatids that must differentiate and individualize into mature sperm. In Drosophila melanogaster, individualization of interconnected spermatids requires the formation of individualization complexes that synchronously move along the sperm bundles. Here, we show that Mob4, a member of the Mps-one binder family, is essential for male fertility but has no detectable role in female fertility. We show that Mob4 is required for proper axonemal structure and its loss leads to male sterility associated with defective spermatid individualization and absence of mature sperm in the seminal vesicles. Transmission electron micrographs of developing spermatids following mob4RNAi revealed expansion of the outer axonemal microtubules such that the 9 doublets no longer remained linked to each other and defective mitochondrial organization. Mob4 is a STRIPAK component, and male fertility is similarly impaired upon depletion of the STRIPAK components, Strip and Cka. Expression of the human Mob4 gene rescues all phenotypes of Drosophila mob4 downregulation, indicating that the gene is evolutionarily and functionally conserved. Together, this suggests that Mob4 contributes to the regulation of the microtubule- and actin-cytoskeleton during spermatogenesis through the conserved STRIPAK complex. Our study advances the understanding of male infertility by uncovering the requirement for Mob4 in sperm individualization.This work was funded by the Algarve 2020 Program, grant number ALG-01-0145-FEDER-030014, cofinanced by FEDER Funds through the Operational Program for Competitiveness Factors—COMPETE 2020 and by national funds through FCT—Foundation for Science and Technology under the Project PTDC/BIA-CEL/30014/2017. IBS was funded by FCT fellowship SFRH/BD/141734/2018. AW acknowledges Cancer Research UK for a PhD studentship (1999-2003). GC was funded by the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR 2020CLZ5XW). DMG acknowledges past grants from CRUK and Wellcome and current support from NIH grant R01NS119614.N
Centrosome function and assembly in animal cells
No full textIt has become clear that the role of centrosomes extends well beyond that of important microtubule organizers. There is increasing evidence that they also function as coordination centres in eukaryotic cells, at which specific cytoplasmic proteins interact at high concentrations and important cell decisions are made. Accordingly, hundreds of proteins are concentrated at centrosomes, including cell cycle regulators, checkpoint proteins and signalling molecules. Nevertheless, several observations have raised the question of whether centrosomes are essential for many cell processes. Recent findings have shed light on the functions of centrosomes in animal cells and on the molecular mechanisms of centrosome assembly, in particular during mitosis. These advances should ultimately allow the in vitro reconstitution of functional centrosomes from their component proteins to unlock the secrets of these enigmatic organelles
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