The centrosomal protein CP190 regulates myosin function during early Drosophila development.

Centrosomes are the main microtubule (MT)-organizing centers in animal cells, but they also influence the actin/myosin cytoskeleton. The Drosophila CP190 protein is nuclear in interphase, interacts with centrosomes during mitosis, and binds to MTs directly in vitro. CP190 has an essential function in the nucleus as a chromatin insulator, but centrosomes and MTs appear unperturbed in Cp190 mutants. Thus, the centrosomal function of CP190, if any, is unclear. Here, we examine the function of CP190 in Cp190 mutant germline clone embryos. Mitosis is not perturbed in these embryos, but they fail in axial expansion, an actin/myosin-dependent process that distributes the nuclei along the anterior-to-posterior axis of the embryo. Myosin organization is disrupted in these embryos, but actin appears unaffected. Moreover, a constitutively activated form of the myosin regulatory light chain can rescue the axial expansion defect in mutant embryos, suggesting that CP190 acts upstream of myosin activation. A CP190 mutant that cannot bind to MTs or centrosomes can rescue the lethality associated with Cp190 mutations, presumably because it retains its nuclear functions, but it cannot rescue the defects in myosin organization in embryos. Thus, CP190 has distinct nuclear and centrosomal functions, and it provides a crucial link between the centrosome/MT and actin/myosin cytoskeletal systems in early embryos.

Kinetochore dynein: its dynamics and role in the transport of the Rough deal checkpoint protein.

We describe the dynamics of kinetochore dynein-dynactin in living Drosophila embryos and examine the effect of mutant dynein on the metaphase checkpoint. A functional conjugate of dynamitin with green fluorescent protein accumulates rapidly at prometaphase kinetochores, and subsequently migrates off kinetochores towards the poles during late prometaphase and metaphase. This behaviour is seen for several metaphase checkpoint proteins, including Rough deal (Rod). In neuroblasts, hypomorphic dynein mutants accumulate in metaphase and block the normal redistribution of Rod from kinetochores to microtubules. By transporting checkpoint proteins away from correctly attached kinetochores, dynein might contribute to shutting off the metaphase checkpoint, allowing anaphase to ensue.

The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore.

The zeste-white 10 (zw10) and rough deal (rod) genes of Drosophila both encode kinetochore components, and mutations in either gene greatly increase the missegregation of sister chromatids during mitosis. Here, we present genetic, cytological and biochemical evidence for a close, evolutionarily conserved relationship between the ROD and ZW10 proteins. We show that the phenotypes caused by disruption of either gene's function are similar in Drosophila and in C. elegans. No additive effects are observed in zw10; rod double null mutants. In flies, the two proteins always colocalize and, moreover, require each other for their recruitment to the mitotic apparatus. The human ROD and ZW10 homologs also colocalize on HeLa cell kinetochores or kinetochore microtubules throughout most but not all of mitosis. Finally, we show that in both Drosophila and human cells, ROD and ZW10 are in fact physically associated, and in Drosophila these proteins are together constituents of a large (700-900 kDa), soluble macromolecular complex.

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton.

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.

Swallowing dynein: a missing link in RNA localization?

Localization of bicoid messenger RNA to the anterior cortex of the developing oocyte is essential for correct anterior-posterior patterning of the Drosophila embryo. It now seems that the Swallow protein functions as an adaptor, bridging bicoid mRNA to dynein, a molecular motor that would transport the complex anteriorly along microtubules.

Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.

The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila.

Mutations in the rough deal (rod) gene of Drosophila greatly increase the missegregation of sister chromatids during mitosis, suggesting a role for this gene product in spindle or kinetochore function. The activity provided by rod also appears to be necessary for the recruitment of two known kinetochore components, Zw10 and cytoplasmic dynein. In this paper we describe the cloning of rough deal and an initial cytological characterization of its product. The Rod protein shares no identifiable structural motif with other known proteins, although apparent homologs exist in the genomes of nematode and man. By immunocytochemistry we show that Rod displays a dynamic intracellular staining pattern, localizing first to kinetochores in prometaphase, but moving to kinetochore microtubules at metaphase. Early in anaphase the protein is once again restricted to the kinetochores, where it persists until the end of telophase. This behavior is in all respects similar to that described for Zw10, and suggests that the proteins function together.

Evidence for redundancy but not trans factor-cis element coevolution in the regulation of Drosophila Yp genes.

In Drosophila melanogaster and the endemic Hawaiian species D. grimshawi three Yolk protein (Yp) genes are expressed in a similar sex- and tissue-specific pattern. In contrast, DNA sequence comparisons of promoter/enhancer regions show low levels of similarity. We tested the functional significance of these observations by transforming D. melanogaster with the genomic region that includes the divergently transcribed D. grimshawi DgYp1 and DgYp2 genes; we found that the introduced genes were expressed in female fat body and in ovaries but not in males. Moreover, we found D. grimshawi proteins in the hemolymph and accumulating in ovaries. Using reporter constructs we showed that the intergenic region from D. grimshawi was sufficient to drive accurate expression, but some low level of ectopic expression was seen in males. Transforming D. melanogaster with constructs bearing deletions within the D. grimshawi intergenic region revealed only subtle effects in the overall level of expression, suggesting a high level of redundancy. Testing mutants in the sex-specific regulator doublesex revealed that it is capable of repressing the DgYp genes in males. Together, these data show that D. melanogaster trans-acting factors can regulate the in vivo pattern of DgYp expression and support the notion of a redundant and complex system of cis-acting elements.

Binuclear Drosophila oocytes: consequences and implications for dorsal-ventral patterning in oogenesis and embryogenesis.

The position of the nucleus along the anterior rim of stage 8 Drosophila oocytes presages the dorsal side of the egg and the developing embryo. In this paper, we address the question of whether the oocyte has a previously determined dorsal side to which the nucleus is drawn, or whether nuclear position randomly determines the dorsal side. To do so, we have taken advantage of a genetic system in which Drosophila oocytes occasionally become binuclear. We find that (i) the two nuclei migrate independently to their respective positions on the anterior rim, sometimes selecting the same site, sometimes not, (ii) the two nuclei are equivalent in their ability to induce a dorsal-ventral pattern in the overlying follicular epithelium, and (iii) at any position around the anterior circumference of the egg chamber the follicle cell sheet is equally responsive to the Gurken signal associated with the oocyte nuclei. These results argue that the dorsal-ventral axis is determined arbitrarily by the randomly selected position of the nucleus on the anterior rim of the oocyte. Some of the binuclear eggs support embryonic development. However, despite the duplication of dorsal chorion structures, the majority of such embryos show normal dorsal-ventral patterning. Thus, processes exist in the ventral follicular epithelium or in the perivitelline space that compensate for the expansion of dorsal follicle cell fates and consequently allow the formation of a normal embryonic axis.

Myosin light chain-activating phosphorylation sites are required for oogenesis in Drosophila.

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.

Drosophila nonmuscle myosin II is required for rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early embryos.

The X-linked Drosophila gene spaghetti squash (sqh) encodes the regulatory light chain of nonmuscle myosin II. To assess the requirement for myosin II in oogenesis and early embryogenesis, we induced homozygous germline clones of the hypomorphic mutation sqh1 in otherwise heterozygous mothers. Developing oocytes in such sqh1 germline clones often failed to attain full size due to a defect in 'dumping', the rapid phase of cytoplasmic transport from nurse cells. In contrast to other dumpless mutants described to date, sqh1 egg chambers showed no evidence of ring canal obstruction, and no obvious alteration in the actin network. However the distribution of myosin II was abnormal. We conclude that the molecular motor responsible for cytoplasmic dumping is supplied largely, if not exclusively, by nurse cell myosin II and we suggest that regulation of myosin activity is one means by which cytoplasmic transport may be controlled during oocyte development. The eggs resulting from sqh1 clones, though smaller than normal, began development but exhibited an early defect in axial migration of cleavage nuclei towards the posterior pole of the embryo, in a similar manner to that seen in early cleavage eggs in which the actin cytoskeleton is disrupted. Thus both nurse cell dumping and axial migration require a maternally supplied myosin II.

Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila.

We describe a new mitotic locus of Drosophila melanogaster required for the progression through mitosis in the syncytial embryo and in late larval development. The locus aar (abnormal anaphase resolution) maps to the cytological interval 85E7-F16 and was identified by two alleles. The aar1 allele causes pupal lethality. Larval neuroblasts show an elevated mitotic index with high chromosome condensation and stretched and lagging chromatids during anaphase. aar2 produces fully viable but sterile females. aar1/aar2 females lay eggs that develop mitotic figures with similar abnormalities to those observed in neuroblasts. Indirect immunofluorescence of these embryos indicates that the centrosome cycle appears normal, although some abnormal spindle microtubules can be seen during mitosis.

Structurally distinct genes for the surface protease of Leishmania mexicana are developmentally regulated.

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed in both the promastigote and the amastigote forms of Leishmania species. In Leishmania mexicana, gp63 exists as a heterogeneous family of proteins that are differentially processed and localized during the 2 developmental stages. In this study we determined the molecular organization of the L. mexicana gp63 gene family, demonstrating that the gp63 genes fall into 3 linked families of tandemly repeated, but structurally distinct, entities designated as C1, C2 and C3. The C1 and C2 gene clusters contain 4-5 copies each, while the C3 gene may be single copy. Whilst promastigotes contain transcripts from all 3 gene classes, the intracellular amastigote only expresses detectable transcript from the C1 gene class. Moreover, the sequence of the C1 genes predicts a unique carboxy terminus substantially different from the GPI anchor addition signal sequence found in other Leishmania spp. and which has characteristics incompatible with substitution with a GPI anchor. These findings have significance for both the diversity of gp63 and for the regulation of tightly clustered, tandem gene arrays.

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila.

Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh1, a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis failure and other defects in sqh1. Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.

rough deal: a gene required for proper mitotic segregation in Drosophila.

We describe a genetic locus rough deal (rod) in Drosophila melanogaster, identified by mutations that interfere with the faithful transmission of chromosomes to daughter cells during mitosis. Five mutant alleles were isolated, each associated with a similar set of mitotic abnormalities in the dividing neuroblasts of homozygous mutant larvae: high frequencies of aneuploid cells and abnormal anaphase figures, in which chromatids may lag, form bridges, or completely fail to separate. Surviving homozygous adults are sterile, and show cuticular defects associated with cell death, i.e., roughened eyes, sparse abdominal bristles, and notched wing margins. The morphological process of spermatogenesis is largely unaffected and motile sperm are produced, but meiocyte aneuploidy is common. The nature of the observed abnormalities in mitotic cells suggests that the reduced fidelity of chromosome transmission to the daughter cells is due to a failure in a mechanism involved in assuring the proper release of sister chromatids.

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage.

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.

Mitosis in Drosophila development.

Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.

Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosomal locus and are constitutively transcribed.

The major surface glycoprotein of Leishmania, gp63, is encoded by a small multi-gene family of tandemly linked genes which map to a single chromosome. For Leishmania major, there are five 3.1 kilobasepair (kb) direct repeat units which include a 1.8-kb open reading frame and a 1.3-kb intergenic or spacer region. In addition, there is a single gene copy linked as a direct repeat but separated from the tandem array of gp63 genes by about 8 kb. The restriction enzyme map of the repeat unit is highly conserved among the gene copies. The regions which flank the tandemly repeated genes diverge outside of the 3.1-kb repeat unit. Transcription of the gp63 gene locus is constitutive as the 3-kb transcript was present in promastigotes as well as in amastigotes. A minor 6-kb gp63 gene transcript was also detected in Northern blot analysis which could signify the transcription of the genes as a polycistronic or multigene precursor RNA.