PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen.

Viruses of the DNA tumor virus family share the ability to transform vertebrate cells through the action of virus-encoded tumor antigens that interfere with normal cell physiology. They accomplish this very efficiently by inhibiting endogenous tumor suppressor proteins that control cell proliferation and apoptosis. Simian virus 40 (SV40) encodes two oncoproteins, large tumor antigen, which directly inhibits the tumor suppressors p53 and Rb, and small tumor antigen (ST), which interferes with serine/threonine protein phosphatase 2A (PP2A). We have constructed a Drosophila model for SV40 ST expression and show that ST induces supernumerary centrosomes, an activity we also demonstrate in human cells. In early Drosophila embryos, ST also caused increased microtubule stability, chromosome segregation errors, defective assembly of actin into cleavage furrows, cleavage failure, a rise in cyclin E levels and embryonic lethality. Using ST mutants and genetic interaction experiments between ST and PP2A subunit mutations, we show that all of these phenotypes are dependent on ST's interaction with PP2A. These analyses demonstrate the validity and utility of Drosophila as a model for viral oncoprotein function in vivo.

Zygotic development without functional mitotic centrosomes.

The centrosome is the dominant microtubule-organizing center in animal cells. At the onset of mitosis, each cell normally has two centrosomes that lie on opposite sides of the nucleus. Centrosomes nucleate the growth of microtubules and orchestrate the efficient assembly of the mitotic spindle. Recent studies in vivo and in vitro have shown that the spindle can form even in the absence of centrosomes and demonstrate that individual cells can divide without this organelle. However, since centrosomes are involved in multiple processes in vivo, including polarized cell divisions, which are an essential developmental mechanism for producing differentiated cell types, it remains to be shown whether or not a complete organism can develop without centrosomes. Here we show that in Drosophila a centrosomin (cnn) null mutant, which fails to assemble fully functional mitotic centrosomes and has few or no detectable astral microtubules, can develop into an adult fly. These results challenge long-held assumptions that the centrosome and the astral microtubules emanating from it are essential for development and are required specifically for spindle orientation during asymmetric cell divisions.

The centrosome in Drosophila oocyte development.

The Drosophila oocyte is a highly specialized cell type whose development utilizes MTOCs in various contexts. Figure 4 (see color insert) summarizes the characteristics of the MTOCs at different stages of oogenesis. Polarized mitoses are required to achieve oocyte determination. In the asymmetric germ-cell divisions that culminate in the egg chamber, the mitotic centrosomes are anchored to the spectrosome or fusome in order to produce the regular branching pattern of the cyst cells. It appears that the primary role of the fusome is to orchestrate the polarity and synchrony of oogenic mitoses. In the absence of fusomes or anchored spindles, the regular interconnected cyst network is lost and the oocyte does not differentiate. It is not known if the spindle itself is asymmetric, or whether either centrosome has equal potential to interact with the fusome. Several models can explain the need for polarized mitoses for oocyte differentiation. In one, an unequal distribution of unknown oocyte differentiation factors occurs from as early as the first cystoblast division. Here, the fusome may be required for the distribution of the factors. In another model, there is a mechanism that measures the number of ring canals in the cell, limiting the choice of oocyte to two potential pro-oocytes. In this model, polarized, synchronous divisions must occur to produce only two cells with the highest number of ring canals. In both of these models the centrosome plays an indirect role. A critical event in the determination of the oocyte is the formation of the MTOC. The oocyte MTOC forms shortly after completion of the germ cell mitoses and establishes a microtubule array along which factors required for oocyte determination are transported. It is unclear how this single MTOC forms in the 16-cell cyst, how the centrosomes become inactivated in the adjoining 15 nurse cells, or why the inactivated centrioles are transported into the oocyte. No molecular components of the MTOC are known except for centrosomin, which accumulates at the MTOC relatively late, at approximately stage 5 or 6 of oogenesis. The MTOC plays a central role in establishing the oocyte's polar coordinates. The oocyte microtubule array is required for the polar localization of axis-determining factors. At midoogenesis the MTOC appears to mediate the reversal of the microtubule array and the migration of the nucleus in the oocyte. The posterior follicle cells signal this reversal after receiving the gurken signal. What changes occur at the MTOC to trigger this cytoskeletal rearrangement? A better understanding of the MTOC's molecular components is necessary before we can begin to unravel the mechanisms underlying these events. The morphology of the MTOC changes after it shifts to the oocyte anterior. Staining with anti-centrosomin antibodies shows that the MTOC changes from discrete nucleus-associated bodies into a broad structure associated with the anterior cortex. The molecular mechanisms underlying this structural rearrangement of the MTOC at midoogenesis are presently unknown. Meiosis I occurs in the absence of centrosomes, but meiosis II spindles are linked by a shared, acentriolar, astral MTOC. The organization of the meiosis I spindle poles requires the NCD motor protein; however, the meiosis I spindle poles are acentriolar and contain no known centrosomal core proteins. The meiosis II astral spindle pole has a unique ring-shaped morphology and contains centrosomal proteins, such as gamma-tubulin. Strong mutations in the maternal gamma Tub37C gene do not block meiosis I, but prevent the progression of meiosis II.

The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions.

A novel DNA-binding protein bound to the mitochondrial inner membrane restores the null mutation of mitochondrial histone Abf2p in Saccharomyces cerevisiae.

The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes.

Drosophila centrosomin protein is required for male meiosis and assembly of the flagellar axoneme.

Centrosomes and microtubules play crucial roles during cell division and differentiation. Spermatogenesis is a useful system for studying centrosomal function since it involves both mitosis and meiosis, and also transformation of the centriole into the sperm basal body. Centrosomin is a protein localized to the mitotic centrosomes in Drosophila melanogaster. We have found a novel isoform of centrosomin expressed during spermatogenesis. Additionally, an anticentrosomin antibody labels both the mitotic and meiotic centrosomes as well as the basal body. Mutational analysis shows that centrosomin is required for spindle organization during meiosis and for organization of the sperm axoneme. These results suggest that centrosomin is a necessary component of the meiotic centrosomes and the spermatid basal body.

SHM1: a multicopy suppressor of a temperature-sensitive null mutation in the HMG1-like abf2 gene.

HM, an HMG1-like mitochondrial DNA-binding protein, is required for maintenance of the yeast mitochondrial genome when cells are grown in glucose. To better understand the role of HM in mitochondria, we have isolated several multicopy suppressors of the temperature-sensitive defect associated with an abf2 null mutation (lacking HM protein). One of these suppressors, SHM1, has been characterized at the molecular level and is described herein. SHM1 encodes a protein (SHM1p) that shares sequence similarity to a family of mitochondrial carrier proteins. On glycerol medium, where mitochondrial function is required for growth, shm1 deletion mutants are able to grow whereas shm1 abf2 double mutants are severely inhibited. These results suggest the SHM1p plays an accessory role to HM in the mitochondrion.

The mitochondrial histone HM: an evolutionary link between bacterial HU and nuclear HMG1 proteins.

The mitochondrial histone HM is a very abundant protein in yeast mitochondria that wraps DNA and activates transcription in vitro and is required within the cell for proper maintenance of the mitochondrial chromosome. HM and the bacterial histone-like protein HU have similar activities in vitro and can substitute for each other in E coli cells and in yeast mitochondria. HM also appears to be functionally homologous to nuclear HMG1 proteins, with which it shares a high degree of sequence homology. We report here the isolation of extragenic suppressors of the yeast HM mutant temperature-sensitive phenotype. We also examined the effects of the lack of HM protein and of respiration deficiency on yeast cells mutant for the NHP6 proteins, the putative yeast nuclear HMG1 homologues.

Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A.

The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype. HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1. Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells). The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation. These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.

Functional complementarity between the HMG1-like yeast mitochondrial histone HM and the bacterial histone-like protein HU.

The mitochondrial histone HM is the major DNA-binding protein in mitochondria and is necessary for maintenance of the mitochondrial genome in the yeast Saccharomyces cerevisiae during growth on fermentable sugars. HM and the Escherichia coli histone-like protein HU have similar activities in vitro, including DNA supercoiling, but share no sequence similarity. We show that HU can functionally complement the respiration deficiency associated with yeast strains lacking HM. Conversely, phenotypes of E. coli cells lacking HU protein, including nucleoid loss and a filamentous cell morphology, were alleviated by expression of HM in these cells. The HU protein of bacteria and the HM protein of mitochondria are therefore functionally complementary in vivo. Functional similarities among HM, HU, and the nuclear HMG1 proteins are implicated and discussed.