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Competing Interests: BD received grants for speeches and advisory boards from Angelini, Astrazeneca, GSK, Novartis, Sanofi and Zambon. BL received fees from Astrazeneca, GSK, Novartis and Sanofi. BC received fees from GSK, Novartis and Sanofi. CL received fees for lectures from Alk Abellò, Astrazeneca, GSK, Novartis and Sanofi. DCE received fees for consultation, lectures and advisory board from Astrazeneca, GSK, Novartis, Regeneron and Sanofi. LMI reveived fees from Chiesi, DMG Firma, GSK, Novartis and Sanofi. MG has received fees from GSK, Novartis and Sanofi. MA received fee for advisory board and speaker for Astra Zeneca, Chiesi, CSL Boerhing, GSK, Novartis, Sanofi, and Takeda. SV partecipated in advisory boards and scientific meetings on behalf of Astrazeneca, Firma, GSK, Novartis and Sanofi. VA received fees for lectures and advisory boards from Astrazeneca, GSK, Novartis, and Sanofi. PP received grants for speeches and expert opinion from GSK, LeoPharma, and Novartis. CFRM received fees from GSK, Novartis and Sanofi. RM and SC are GSK full time employes. Macchi A, ML, NS, PG and PL declare to have no conflicts of interest.

Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a pericentriolar material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4's localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.

Centrioles are found in the centrosome core and, as basal bodies, at the base of cilia and flagella. Centriole assembly and duplication is controlled by Polo-like-kinase 4 (Plk4): these processes fail if Plk4 is downregulated and are promoted by Plk4 overexpression. Here we show that the centriolar protein Asterless (Asl; human orthologue CEP152) provides a conserved molecular platform, the amino terminus of which interacts with the cryptic Polo box of Plk4 whereas the carboxy terminus interacts with the centriolar protein Sas-4 (CPAP in humans). Drosophila Asl and human CEP152 are required for the centrosomal loading of Plk4 in Drosophila and CPAP in human cells, respectively. Depletion of Asl or CEP152 caused failure of centrosome duplication; their overexpression led to de novo centriole formation in Drosophila eggs, duplication of free centrosomes in Drosophila embryos, and centrosome amplification in cultured Drosophila and human cells. Overexpression of a Plk4-binding-deficient mutant of Asl prevented centriole duplication in cultured cells and embryos. However, this mutant protein was able to promote microtubule organizing centre (MTOC) formation in both embryos and oocytes. Such MTOCs had pericentriolar material and the centriolar protein Sas-4, but no centrioles at their core. Formation of such acentriolar MTOCs could be phenocopied by overexpression of Sas-4 in oocytes or embryos. Our findings identify independent functions for Asl as a scaffold for Plk4 and Sas-4 that facilitates self-assembly and duplication of the centriole and organization of pericentriolar material.

Centrioles are essential for the formation of microtubule-derived structures, including cilia and centrosomes. Abnormalities in centrosome number and structure occur in many cancers and are associated with genomic instability. In most dividing animal cells, centriole formation is coordinated with DNA replication and is highly regulated such that only one daughter centriole forms close to each mother centriole. Centriole formation is triggered and dependent on a conserved kinase, SAK/PLK4. Downregulation and overexpression of SAK/PLK4 is associated with cancer in humans, mice, and flies. Here we show that centrosome amplification is normally inhibited by degradation of SAK/PK4 degradation, mediated by the SCF/Slimb ubiquitin ligase. This complex physically interacts with SAK/PLK4, and in its absence, SAK/PLK4 accumulates, leading to the striking formation of multiple daughter centrioles surrounding each mother. This interaction is mediated via a conserved Slimb binding motif in SAK/PLK4, mutations of which leads to centrosome amplification. This regulation is likely to be conserved, because knockout of the ortholog of Slimb, beta-Trcp1 in mice, also leads to centrosome amplification. Because the SCF/beta-Trcp complex plays an important role in cell-cycle progression, our results lead to new understanding of the control of centrosome number and how it may go awry in human disease.

Centrioles are essential for the formation of cilia, flagella and centrosome organization. Abnormalities in centrosome structure and number in many cancers can be associated with aberrant cell division and genomic instability.(1,2) Canonical centriole duplication occurs in coordination with the cell division cycle, such that a single new "daughter" centriole arises next to each "mother" centriole. If destroyed, or eliminated during development, centrioles can form de novo.(3-5) Here we discuss our recent data demonstrating a molecular pathway that operates in both de novo and canonical centriole biogenesis involving SAK/PLK4, SAS-6 and SAS-4.(6) We showed that centriole biogenesis is a self-assembly process locally triggered by high SAK/PLK4 activity that may or not be associated with an existing centriole. SAS-6 acts downstream of SAK/PLK4 to organize nine precentriolar units, which we call here enatosomes, fitting together laterally and longitudinally, specifying a tube-like centriole precursor.(7,8) The identification of mutants impaired in centriole biogenesis has permitted the study of the physiological consequences of their absence in the whole organism. In Drosophila, centrioles are not necessary for somatic cell divisions.(9,10) However, we show here that mitotic abnormalities arise in syncytial SAK/PLK4-derived mutant embryos resulting in lethality. Moreover male meiosis fails in both SAK/PLK4 and DSAS-4 mutant spermatids that have no centrioles. These results show diversity in the need for centrioles in cell division. This suggests that tissue specific constraints selected for different contributions of centrosome-independent and dependent mechanisms in spindle function. This heterogeneity should be taken into account both in reaching an understanding of spindle function and when designing drugs that target cell division.

Centrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia [1]. Abnormalities in centrosome structure and number occur in many cancers [1, 2]. Despite its importance, very little is known about centriole biogenesis. Recent studies in C. elegans have highlighted a group of molecules necessary for centriole assembly [1, 3]. ZYG-1 kinase recruits a complex of two coiled-coil proteins, SAS-6 and SAS-5, which are necessary to form the C. elegans centriolar tube, a scaffold in centriole formation [4, 5]. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube [4, 5]. Here we show that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos led to the de novo formation of multiple microtubule-organizing centers (MTOCs). Strikingly, the center of these MTOCs did not contain centrioles, as described previously for SAK/PLK4 overexpression [6]. Instead, tube-like structures were present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 [5]. In DSAS-6 loss-of-function mutants, centrioles failed to close and to elongate the structure along all axes of the 9-fold symmetry, suggesting modularity in centriole assembly. We propose that the tube is built from nine subunits fitting together laterally and longitudinally in a modular and sequential fashion, like pieces of a layered "hollow" cake.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids., (Copyright 1999 John Wiley & Sons, Ltd.)

Using transmission electron microscopy, immuno-electron microscopy, and biochemical techniques such as 2-D electrophoresis and immunoblotting, actin was found in all biological stages of the microsporidia Encephalitozoon hellem and Encephalitozoon cuniculi.

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.

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.

The origin of the zygotic centrosome is an important step in developmental biology. It is generally thought that sperm at fertilization plays a central role in forming the functional centrosome which subsequently organizes the first mitotic spindle. However, this view is not applicable in the case of parthenogenetic eggs which develop without the sperm contribution. To clarify the problem of the origin of the zygotic centrosome during parthenogenetic development, we studied a hymenopteran, Muscidifurax uniraptor. Antitubulin antibody revealed that after activation several asters assembled in the egg cytoplasm. The number of asters varied in relation to the cell cycle. They became visible from anaphase of the first meiotic division and increased in number as meiosis progressed, reaching a maximum at the first mitosis. From anaphase-telophase of the first mitosis they decreased in number and were no longer found during the third mitotic division. To elucidate the nature of these asters we performed an ultrastructural study with transmission electron microscopy and immunofluorescence with antibodies against anti-gamma-tubulin and CP190. In this way we showed the presence in these asters of centrosomal components and centrioles. Our observations suggest that the cytoplasm of Muscidifurax eggs contains a pool of inactive centrosomal precursor proteins becoming able to nucleate microtubules into well-defined asters containing centrioles after activation., (Copyright 1998 Academic Press.)

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.

We have used immunofluorescence and electron microscopy to examine centrosome dynamics during the first postblastodermic mitoses in the Drosophila embryo. The centrosomal material, as recognized by antibodies against CP190 and gamma-tubulin, does not show the typical shape changes observed in syncytial embryos, but remains compact throughout mitosis. Centrioles, however, behave as during the syncytial mitoses, with each daughter cell inheriting two separated centrioles at the end of telophase. During interphase in epithelial cells that have a distinct G1 phase, two isolated centrioles are found, suggesting that the separation of sister centrioles is tightly coupled to a mitotic oscillator in both the "abbreviated" and the "complete" embryonic division cycles. The centrioles of the Drosophila embryo sharply differed from the sperm basal body, having a cartwheel structure with nine microtubular doublets and a central tubule. This "immature" centriolar morphology was shown to persist throughout embryonic development, clearly demonstrating that these centrioles are able to replicate despite their apparently neotenic structure.

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.

Incubation of early Drosophila embryos with low concentrations of taxol (2.3 microM) revealed a pattern of microtubule assembly that was cell-cycle dependent. Microtubule bundling was observed during the pronuclear stage after resumption of meiosis, whereas at the onset of the first mitosis the microtubules organized in astral arrays. Taxol treatment showed differential microtubule assembly properties of the egg cytoplasm. The preferential assembly site for taxol-induced asters was the ventral cortex; in the dorsal cortex only microtubule bundling occurred. This dorsal-ventral heterogeneity of the ege cortex persisted until the third or fourth nuclear cycle. Microtubules did not organize in astral arrays in the inner cytoplasm, but only in mitotic spindles. CP190 and gamma-tubulin, usually found in the centrosome of the early Drosophila embryo, were absent in taxol-induced asters. These observations suggest that the mechanism driving the assembly of taxol-induced asters is not centrosome dependent in the early Drosophila embryo.

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.

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.

Immunofluorescence microscopic observations indicated that a monoclonal antibody, Vmp 18, raised against the peptide 199-208 of murine interleukin 1 alpha, cross-reacted with an antigenic determinant of Drosophila thorax muscles. Immunoelectron microscopic analysis showed that the gold particles were mainly localized in the Z-line which is the attachment site of thin filaments from adjacent sarcomeres. On the contrary, the antibody failed to mark the Z-line in vertebrate skeletal muscle. A Western blot of total protein extract from Drosophila thorax muscles bound a protein of 43 kDa. Our observations suggest that the Vmp 18 antibody could contribute to clarify the composition of the Z-line in insect's flight muscles.

Incubation with the Bcl-2 antibody before fixation of tissues allowed good localization of the antigenic determinant. We showed that the Bcl-2 gene product in centroblastic-centrocytic lymphoma is mainly localized on the outer mitochondrial membrane and, to a lesser degree, on the nuclear envelope. No significant staining was found in other cytoplasmic domains. Careful examination also revealed that gold particles did not recognize an integral membrane epitope, but an antigenic determinant localized at a short distance from the cytoplasmic side of the membrane itself. This observation suggests that, by interacting with other cytoplasmic proteins, Bcl-2 plays some role in the cytoplasmic machinery involved in the regulation of programmed cell death.

Whole-mounts of Drosophila embryos were stained with the monoclonal antibody Vmp 18, raised against the peptide 199-208 of murine interleukin 1/ alpha. Immunofluorescence observations showed that the antibody cross-reacted with an antigenic determinant that changed in localization during Drosophila development. In syncytial Drosophila embryos, the antibody recognized an epitope localized on the nuclear envelope throughout mitotic division. As cellularization occurred, the fluorescence was mainly concentrated in the apical region of the blastoderm cells. Western blot analysis of whole Drosophila embryo extracts showed that the antibody recognized a 60-kDa protein in syncytial embryos and during germ band elongation. This suggests that the 60-kDa antigen undergoes dynamic redistribution during embryogenesis.

Early in development the precursors of germ cells in Drosophila migrate at the posterior pole of the embryo and translocate to the bottom of the developing posterior midgut primordium. At the end of germ band elongation the pole cells cross the gut wall to enter in association with the gonadal mesoderm. We used laser scanning confocal microscopy on whole-mount Rh-phalloidin-stained embryos and transmission electron microscopy to investigate how pole cells cross the epithelial wall of the posterior midgut primordium. Our results suggest that pole cells leave the midgut sac by traveling through the intercellular spaces of the epithelium. During this process the epithelial cells at the bottom of the posterior midgut primordium are greatly deformed, but their junctional complexes do not completely release, avoiding breaks in the epithelial wall.

The distribution of microfilaments in Drosophila egg chambers stained with rhodamine (Rh)-conjugated phalloidin was studied by laser scanning confocal microscopy and transmission electron microscopy. These techniques revealed new details in the pattern of microfilament localization. We observed in stage 1-3 egg chambers accumulation of filamentous actin in the oocyte cytoplasm between the ring canals connecting the oocyte with adjacent nurse cells. Starting from stages 6-7 short microfilament bundles arranged in basket-like structures were associated with the side of the ring canals facing the nurse cell cytoplasm. We also observed a dramatic decrease in the actin network associated with the cortex of the oocyte in stage 10. During stage 10B the nurse cell cytoplasm was crossed by radial actin bundles that showed a sarcomeric-like cross striation after Rh-phalloidin staining. The ring canals also did not uniformly stain but showed a punctate labeling. The implications of the actin cytoskeleton during oocyte growth are discussed.

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.

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.

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.

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.

Microfilament integrity is needed to maintain the regular arrangement of the spindle microtubules and to guarantee the normal progression of the last syncytial mitoses in Drosophila embryo. To investigate when and how microfilaments participate in this process, we incubated permeabilized embryos with the inhibitor of actin polymerization, cytochalasin B, at different times during the nuclear cycle. Our results suggest that the correct microfilament distribution is only required for the appropriate segregation of nuclei during the 11th, 12th and 13th syncytial mitoses rather than during the 10th mitosis when the spindles are too far apart to interact. When cytochalasin B treatment was performed during the last syncytial mitoses many spindles fuse among them and the regular mitotic progression is perturbed.

Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle.

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.

Centriole and centrosome cycles were examined by indirect immunofluorescence and electron microscopy techniques in the early Drosophila embryo. The centrosomes, which are already divided at interphase, appear as compact spheres during prophase and metaphase, expand and flatten from anaphase to telophase and split into two units in late telophase. Centriole separation starts in late metaphase, becomes evident in anaphase and increases during telophase. Procentrioles appear during the following interphase.