Centriole symmetry: a big tale from small organisms.

Centrioles are microtubule-based cylindrical organelles with a 9-fold symmetry. They are essential for axoneme formation in cilia and flagella and for centrosome organization. In the basal hexapods Acerentomon microrhinus, we discovered unusually large centrioles composed of 14 doublet microtubules that serve as templates for cilia and flagella and organize mitotic and meiotic spindles. These observations challenge the long-standing view that centriole symmetry is highly conserved among eukaryotes. Strikingly, daughter centrioles contain a transient cartwheel that is lost after maturation. The length of radial spokes is like that found in 9-fold cartwheels, whereas the diameter of the hub varies according to the dimensions of the centriole cylinder. This suggests that the hub may dictate the master plan for centriole geometry. Finally, the finding that 14-doublet centrioles arise from 9-doublet mothers points to an alternative model for centriole assembly.

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia.

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.

Drosophila polo kinase is required for cytokinesis.

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.

Nup154, a new Drosophila gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene.

The Nup154 gene of Drosophila encodes a protein showing similarity with known nucleoporins: rat Nup155 and yeast Nup170 and Nup157. Hypomorphic mutant alleles of Nup154 affected female and male fertility, allowing investigation of the gene function in various steps of oogenesis and spermatogenesis. Nup154 was required in testes for cyst formation, control of spermatocyte proliferation and meiotic progression. In ovaries, Nup154 was essential for egg chamber development and oocyte growth. In both the male and female germ line, as well as in several other cell types, the Nup154 protein was detected at the nuclear membrane, but was also present inside the nucleus. Intranuclear localization has not previously been described for rat Nup155 or yeast Nup170 and Nup157. In mutant egg chambers the Nup154 protein accumulated in the cytoplasm, while it was only barely detected at the nuclear envelopes. FG repeats containing nucleoporins detected with mAb414 antibody were also mislocalized to a certain extent in Nup154 mutant alleles. This suggests that Nup154 could be required for localizing other nucleoporins within the nuclear pore complex, as previously demonstrated for the yeast Nup170. On the other hand, no evident defects in lamin localization were observed, indicating that Nup155 mutations did not affect the overall integrity of the nuclear envelope. However, ultrastructural analyses revealed that in mutant cells the morphology of the nuclear envelope was altered near the nuclear pore complexes. Finally, the multiplicity of phenotypes observed in Nup154 mutant alleles suggests that this gene plays a crucial role in cell physiology.

SAK/PLK4 is required for centriole duplication and flagella development.

BACKGROUND: SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. RESULTS: Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. CONCLUSIONS: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.

Assembly of the zygotic centrosome in the fertilized Drosophila egg.

Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein (Whitfield et al. (1988) J. Cell Sci. 89, 467-480; Whitfield et al. (1995) J. Cell Sci. 108, 3377-3387). The CP190 protein was detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids matured, this labelling gradually disappeared and was no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin was also found in association with the posterior end of the sperm nucleus during spermiogenesis, but was not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 was not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles were associated with microtubules of the sperm aster from anaphase I to anaphase II. These particles disappeared during early telophase II and only one pair of CP190-positive spots remained visible at the microtubule focus of the sperm aster. These spots were associated with one aster through telophase, and then moved away to form two smaller asters from which the first mitotic spindle was organized. Colchicine treatment suggested that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen was prevented by incubation of the permeabilized zygote in 20 mM EDTA.

gamma-Tubulin is transiently associated with the Drosophila oocyte meiotic apparatus.

Evidence of a distinct microtubule organizing center in the meiotic apparatus of the fertilized Drosophila egg is provided by means of specific antibodies. This center contained gamma-tubulin and CP190 antigens and nucleated a transient array of radial microtubules. When the eggs were incubated with the microtubule-depolymerizing drug colchicine, gamma-tubulin became undetectable in correspondence with the meiotic chromosomes, whereas it was visible near the sperm nucleus. Since the main difference between male and female microtubule organizing centers was the presence/absence of the centrioles, we propose that these organelles were mainly involved in the spatial organization of the microtubule nucleating material.

Primordial germ cell migration in the Ceratitis capitata embryo.

In this study we followed the behavior of germ cell precursors in the early embryo of the dipteran Ceratitis capitata using conventional fluorescence, laser scanning confocal and transmissiom electron microscopies. During cellularization the pole cells formed a cluster which lodged in a roundish break in the blastoderm at the posterior pole of the embryo. When gastrulation began, the pole cells shifted dorsally and during elongation of the germ band moved into the posterior midgut primordium. Pole cell morphology suggested that these cells were motile until the early stages of development.

Spatial organization of microtubules and microfilaments in larval and adult salivary glands of Drosophila melanogaster.

We examined the distribution of microtubules and microfilaments by conventional fluorescence microscopy and laser scanning confocal microscopy in larval and adult salivary glands of Drosophila melanogaster. The cells of the larval salivary gland epithelium were characterized by the same spatial distribution of microfilaments, whereas microfilament localization was more complex in adult salivary glands, showing some regional differentiation. Microtubules distributed throughout the cell cytoplasm of the larval salivary glands, whereas in adult glands they were mostly observed in the basal or apical cytoplasm of the cells. These observations were related to the secretory process and the mechanism of saliva discharge.

Revisiting the role of the mother centriole in centriole biogenesis.

Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.

Microtubule distribution reveals superficial metameric patterns in the early Drosophila embryo.

Microtubule distribution was examined in whole mounts of Drosophila embryos from the cellularization of the syncytial blastoderm (stage 6) to the completion of the gastrulation (stage 7) by fluorescence microscopy. During ventral furrow formation, the fluorescence of tubulin network was not uniform, but disposed in zebra stripes. Antibodies against alpha-tubulin showed 14 alternating pairs of darker and brighter transverse areas. The possible significance of this pattern is discussed.

Centrosome splitting during nuclear elongation in the Drosophila embryo.

In the early Drosophila embryo, nuclear elongation occurs during cellularization of the syncytial blastoderm. This process is closely related to the presence of microtubular bundles forming a basket-like structure surrounding the nuclei. In immunofluorescence observations with antibodies against alpha-tubulin, the microtubules appear to radiate from two bright foci widely separated from each other. We used electron microscopy to show that these foci are true centrosomes constituted by daughter and parent centrioles orthogonally disposed and surrounded by pericentriolar electrondense material. The centrosomes may be observed in the apical region of the blastoderm cells from the beginning of cellularization until the reestablishment of the first postblastodermic mitosis, when they organize the spindle poles. Until this time the dimensions of the procentrioles remain unchanged. The significance of these results is discussed in relation to the known behavior of centrioles in the cell cycle.

Abnormal centrosomes in cold-treated Drosophila embryos.

In this study we examine the effect on the centrosomes of cold treatment of early Drosophila embryos. Prolonged cold treatment during the mitotic divisions which lead to the formation of the blastoderm causes arrest at metaphase of the nuclear divisions. When examined with immunofluorescence microscopy the mitotic spindles show marked pole splitting with the formation of supernumerary and irregularly sized centers, all able to nucleate microtubules. In embryos recovered for longer periods the additional organizing centers become ring-shaped and lose their nucleating properties. Cold treatment of embryos during the cellularization of the blastoderm results in marked fragmentation of the centrosomes, but nucleating capacity is preserved. Sometimes the centrioles come away from the pericentriolar material and their structure is seen to be modified.

Abnormal behavior of the yolk centrosomes during early embryogenesis of Drosophila melanogaster.

After the 10th nuclear cycle the yolk centrosomes follow an irregular pathway. Unlike the somatic centrosomes, which move to the opposite poles of the nuclei to form the bipolar spindles, the yolk centrosomes remain as pairs at one pole of the yolk nuclei or shift feebly and nucleate irregular spindles, most of which have only one main pole. The yolk centrosomes are no longer observed near the yolk nuclei, but progressively move away into the surrounding cytoplasm. Despite the irregular behavior of the centrosomes and although the yolk nuclei cease to divide, the yolk centrosome duplication cycle continues. The early development of Drosophila thus provides an excellent natural system for the study of the uncoupling of the nuclear and centrosomal cycles.

Surface cap modifications in cold-treated Drosophila melanogaster embryos.

When early Drosophila embryos were allowed to develop at 0 degree C, several abnormalities in the surface cap organization were observed. Scanning electron microscopy showed that exposure to cold mainly lead to the deformation of the cortical caps and to their partial fusion with adjacent caps. The process of cellularization was presumably affected and large uncellularized areas were observed. Rhodamine-phalloidin staining showed that cap deformation was closely related to the altered microfilament distribution, which was presumably responsible for the failure of large syncytial areas to cellularize. During the process of cellularization, F-actin localization did not depend on the microtubules forming the baskets around the elongating nuclei, but was related to the subpopulation of microtubules radiating from the centrosomes toward the plasma membrane. Only these microtubules seemed to be affected by cold treatment.

Involvement of microtubules and microfilaments in centrosome dynamics during the syncytial mitoses of the early Drosophila embryo.

To examine the role of microfilaments and microtubules in centrosome dynamics we exposed Drosophila embryos to culture medium containing cytochalasin B and to low temperature. The results show that the splitting of the centrosomal material does not occur when the embryos are treated with cytochalasin before centrosome duplication at late telophase. The fragmentation of the centrosomal material, caused by cold exposure, is also prevented by cytochalasin incubation. These results indicate that both microtubules and microfilaments may be involved in determining centrosome shape during the syncytial mitoses which lead to the formation of the blastoderm in early Drosophila embryos.

Fertilization in Drosophila melanogaster: centrosome inheritance and organization of the first mitotic spindle.

Microtubule, chromatin, centrosome, and nuclear envelope configurations during the first division of the Drosophila melanogaster zygote were analyzed in order to investigate the organization of the first cleavage spindle and the origin of the functional centrosome. After pronuclear apposition the parental complements congress at the equatorial plane of the metaphase spindle. The chromatids, however, seem to move to the poles in two separate groups in each half spindle, mingling together during telophase, before the formation of the daughter nuclei. The spatial separation of parental complements during the first mitosis is also supported by the behavior of the nuclear envelope of female and male pronuclei. A low frequency of polyspermy is also observed during fertilization in D. melanogaster.

A monoclonal antibody recognizing a common antigen on Drosophila embryos and human fibroblasts.

We used a monoclonal antibody specific for vimentin from human fibroblasts to stain whole mounts of Drosophila embryos. In immunofluorescence observations this antibody cross-reacts with an antigenic determinant localized throughout mitosis at the nuclear boundary. Double fluorescence observations with the Rb188 antibody that specifically recognizes a centrosomal protein of the Drosophila embryo [Whitfield et al., 1988] showed that the anti-vimentin antibody cross-reacts with an antigen localized in the centrosomal region.

Distribution of a nuclear envelope antigen during the syncytial mitoses of the early Drosophila embryo as revealed by laser scanning confocal microscopy.

The changing distribution of a nuclear envelope antigen recognized by a monoclonal antibody raised against human fibroblast vimentin during the syncytial mitoses of the Drosophila embryo has been studied with a confocal laser scanning microscope. The antigen appears very early as irregular aggregates in the peripheral cytoplasm of the preblastoderm embryo. As the first nuclei reach the periplasm the antigen is localized on the nuclear envelope and the cytoplasmic staining decreases. In addition to the perinuclear labeling we observed intense midzone and polar staining during the mitotic cycle. A possible relationship between polar localization of the antigen and centrosome position is discussed.