The COP9 signalosome is essential for development of Drosophila melanogaster.

The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.     

Mitochondrial single-stranded DNA-binding protein is required for mitochondrial DNA replication and development in Drosophila melanogaster

The discovery that several inherited human diseases are caused by mtDNA depletion has led to an increased interest in the replication and maintenance of mtDNA. We have isolated a new mutant in the lopo (low power) gene from Drosophila melanogaster affecting the mitochondrial single-stranded DNA-binding protein (mtSSB), which is one of the key components in mtDNA replication and maintenance. lopo(1) mutants die late in the third instar before completion of metamorphosis because of a failure in cell proliferation. Molecular, histochemical, and physiological experiments show a drastic decrease in mtDNA content that is coupled with the loss of respiration in these mutants. However, the number and morphology of mitochondria are not greatly affected. Immunocytochemical analysis shows that mtSSB is expressed in all tissues but is highly enriched in proliferating tissues and in the developing oocyte. lopo(1) is the first mtSSB mutant in higher eukaryotes, and its analysis demonstrates the essential function of this gene in development, providing an excellent model to study mitochondrial biogenesis in animals.     

The splicing factor Prp31 is essential for photoreceptor development in Drosophila

Retinitis pigmentosa is a leading cause of blindness and a progressive retinal disorder, affecting millions of people worldwide. This disease is characterized by photoreceptor degeneration, eventually leading to complete blindness. Autosomal dominant (adRP) has been associated with mutations in at least four ubiquitously expressed genes encoding pre-mRNA splicing factors—Prp3, Prp8, Prp31 and PAP1. Biological function of adRP-associated splicing factor genes and molecular mechanisms by which mutations in these genes cause cell-type specific photoreceptor degeneration in humans remain to be elucidated. To investigate the in vivo function of these adRP-associated splicing factor genes, we examined Drosophila in which expression of fly Prp31 homolog was down-regulated. Sequence analyses show that CG6876 is the likely candidate of Drosophila melanogaster Prp31 homolog (DmPrp31). Predicted peptide sequence for CG6876 shows 57% similarity to the Homo sapiens Prp31 protein (HsPrp31). Reduction of the endogenous Prp31 by RNAi-mediated knockdown specifically in the eye leads to reduction of eye size or complete absence of eyes with remarkable features of photoreceptor degeneration and recapitulates the bimodal expressivity of human Prp31 mutations in adRP patients. Such transgenic DmPrp31RNAi flies provide a useful tool for identifying genetic modifiers or interacting genes for Prp31. Expression of the human Prp31 in these animals leads to a partial rescue of the eye phenotype. Our results indicate that the Drosophila CG6876 is the fly ortholog of mammalian Prp31 gene.     

Nir2, a human homolog of Drosophila melanogaster retinal degeneration B protein,is essential for cytokinesis

Cytokinesis, the final stage of eukaryotic cell division, ensures the production of two daughter cells. It requires fine coordination between the plasma membrane and cytoskeletal networks, and it is known to be regulated by several intracellular proteins, including the small GTPase Rho and its effectors. In this study we provide evidence that the protein Nir2 is essential for cytokinesis. Microinjection of anti-Nir2 antibodies into interphase cells blocks cytokinesis, as it results in the production of multinucleate cells. Immunolocalization studies revealed that Nir2 is mainly localized in the Golgi apparatus in interphase cells, but it is recruited to the cleavage furrow and the midbody during cytokinesis. Nir2 colocalizes with the small GTPase RhoA in the cleavage furrow and the midbody, and it associates with RhoA in mitotic cells. Its N-terminal region, which contains a phosphatidylinositol transfer domain and a novel Rho-inhibitory domain (Rid), is required for normal cytokinesis, as overexpression of an N-terminal-truncated mutant blocks cytokinesis completion. Time-lapse videomicroscopy revealed that this mutant normally initiates cytokinesis but fails to complete it, due to cleavage furrow regression, while Rid markedly affects cytokinesis due to abnormal contractility. Rid-expressing cells exhibit aberrant ingression and ectopic cleavage sites; the cells fail to segregate into daughter cells and they form a long unseparated bridge-like cytoplasmic structure. These results provide new insight into the cellular functions of Nir2 and introduce it as a novel regulator of cytokinesis.     

The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.     

Talin is essential for integrin function in Drosophila

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.     

DEAF-1 function is essential for the early embryonic development of Drosophila

The Drosophila protein DEAF-1 is a sequence-specific DNA binding protein that was isolated as a putative cofactor of the Hox protein Deformed (Dfd). In this study, we analyze the effects of loss or gain of DEAF-1 function on Drosophila development. Maternal/zygotic mutations of DEAF-1 largely result in early embryonic arrest prior to the expression of zygotic segmentation genes, although a few embryos develop into larvae with segmentation defects of variable severity. Overexpression of DEAF-1 protein in embryos can induce defects in migration/closure of the dorsal epidermis, and overexpression in adult primordia can strongly disrupt the development of eye or wing. The DEAF-1 protein associates with many discrete sites on polytene chromosomes, suggesting that DEAF-1 is a rather general regulator of gene expression.     

Heat shock transcription factor 2 is not essential for embryonic development,fertility, or adult cognitive and psychomotor function in mice

Members of the heat shock factor (HSF) family are evolutionarily conserved regulators that share a highly homologous DNA-binding domain. In mammals, HSF1 is the main factor controlling the stress-inducible expression of Hsp genes while the functions of HSF2 and HSF4 are less clear. Based on its developmental profile of expression, it was hypothesized that HSF2 may play an essential role in brain and heart development, spermatogenesis, and erythroid differentiation. To directly assess this hypothesis and better understand the underlying mechanisms that require HSF2, we generated Hsf2 knockout mice. Here, we report that Hsf2(-/-) mice are viable and fertile and exhibit normal life span and behavioral functions. We conclude that HSF2, most probably because its physiological roles are integrated into a redundant network of gene regulation and function, is dispensable for normal development, fertility, and postnatal psychomotor function.