Neville Cobbe

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis

BACKGROUND Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. We show that CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. We analysed the consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, we discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes were still able to compact, paving the way for the identification of further components or activities required for this essential process.

Diverse mitotic and interphase functions of condensins in Drosophila

The condensin complex has been implicated in the higher-order organization of mitotic chromosomes in a host of model eukaryotes from yeasts to flies and vertebrates. Although chromosomes paradoxically appear to condense in condensin mutants, chromatids are not properly resolved, resulting in chromosome segregation defects during anaphase. We have examined the role of different condensin complex components in interphase chromatin function by examining the effects of various condensin mutations on position-effect variegation in Drosophila melanogaster. Surprisingly, most mutations affecting condensin proteins were often found to result in strong enhancement of variegation in contrast to what might be expected for proteins believed to compact the genome. This suggests either that the role of condensin proteins in interphase differs from their expected role in mitosis or that the way we envision condensins activity needs to be modified to accommodate alternative possibilities.

The evolution of immune-related genes from disease carrying mosquitoes: diversity in a peptidoglycan- and a thioester-recognizing protein

Adaptive polymorphism may be common in immune system genes as co‐evolutionary interactions foster diversity; either through ongoing positive selection (arms races), or balancing selection. DNA sequence diversity in two putative immune system genes was examined in species of the genus Anopheles and from Aedes aegypti. For one gene, encoding the peptidoglycan recognizing protein PGRPLB, there was evidence of purifying selection, suggesting that selection acts to eliminate sequence variation. For another gene, encoding the thioester‐containing protein TEP3, higher levels of amino acid replacement were found than would be expected under neutral models of evolution – an indication that this gene has been subject to repeated bouts of positive selection.

The conserved metalloprotease invadolysin localizes to the surface of lipid droplets

Invadolysin is a metalloprotease conserved in many different organisms, previously shown to be essential in Drosophila with roles in cell division and cell migration. The gene seems to be ubiquitously expressed and four distinct splice variants have been identified in human cells but not in most other species examined. Immunofluorescent detection of human invadolysin in cultured cells reveals the protein to be associated with the surface of lipid droplets. By means of subcellular fractionation, we have independently confirmed the association of invadolysin with lipid droplets. We thus identify invadolysin as the first metalloprotease located on these dynamic organelles. In addition, analysis of larval fat-body morphological appearance and triglyceride levels in the Drosophila invadolysin mutant suggests that invadolysin plays a role in lipid storage or metabolism.

Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling.

Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor–target of rapamycin (InR–TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR–TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR–TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR–TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling.

Why the apparent haste to clone humans

The recent desperation to clone human embryos may be seriously undermining accepted ethical principles of medical research, with potentially profound wider consequences

Drosophila as a Potential Model for Ocular Tumors

Drosophila has made many contributions to our understanding of cancer genes and mechanisms that have subsequently been validated in mammals. Despite anatomical differences between fly and human eyes, flies offer a tractable genetic model in which to dissect the functional importance of genetic lesions found to be affected in human ocular tumors. Here, we discuss different approaches for using Drosophila as a model for ocular cancer and how studies on ocular cancer genes in flies have begun to reveal potential strategies for therapeutic intervention. We also discuss recent developments in the use of Drosophila for drug discovery, which is coming to the fore as Drosophila models are becoming tailored to study tumor types found in the clinic.

Interspecies Mixtures and the Status of Humanity

What does it mean to be a human? Could the mixing of human and nonhuman materials threaten human identity and, if so, how might this happen? This chapter explores such questions in the light of current biological understanding, discussing various features of human life that allegedly mark it off from non-human life, ultimately concluding that any associated characteristics need to be viewed in a holistic manner. The scientific rationale underlying research with various entities that have latterly prompted the greatest controversy is then analyzed in relation to contemporary claims regarding such proposals. Finally, possible approaches towards the ethical evaluation of research involving novel interspecies combinations are presented.

Creation of Human–Animal Entities for Translational Stem Cell Research: Scientific Explanation of Issues That Are Often Confused

In keeping with the Nuremberg Code and the Declaration of Helsinki, the novel use of certain stem cells in patient treatments is likely to require prior testing in animals in order to minimize risks. When human cells are combined with those of other animals in a living creature, this generates something referred to as a chimera. However, a chimera is only one example of the various possible types of interspecies entities that have been used in biological research. How exactly might these different entities be used in translating basic stem cell research towards clinical therapies? Could the mixing of human and nonhuman materials threaten human identity and, if so, how might this happen? This chapter explores such questions in the light of current biological understanding.