Nuclear entry of the Drosophila melanogaster nuclear lamina protein YA correlates with developmentally regulated changes in its phosphorylation state.

The Drosophila melanogaster YA protein is a maternally provided nuclear lamina component that is essential during the transition from meiosis to mitosis at the beginning of embryogenesis. Localization of YA to the nuclear envelope is required for its function; this localization is cell cycle-dependent during embryogenesis. Here we show that the ability of YA to enter nuclei is modulated during development. In developing egg chambers, YA protein is made but excluded from nuclei of nurse cells and oocytes; upon egg activation, YA acquires the ability to enter nuclei and becomes incorporated into the nuclear lamina in unfertilized eggs and embryos. This localization switch correlates with changes in the phosphorylation state of YA. YA in ovaries is hyperphosphorylated relative to YA in unfertilized eggs and embryos. Through site-directed mutagenesis, we identified 443T, a potential phosphorylation site for both cyclin-dependent protein kinase and mitogen-activated-protein kinase, as one of the sites likely involved in this developmental control. Our results suggest that phosphorylation plays a role in modulating the localization of YA during development. A model for developmental regulation of the nuclear entry of YA is proposed and implications for understanding Drosophila egg activation are discussed.     

Interactions among Drosophila Nuclear Envelope Proteins Lamin, Otefin, and YA

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH₂-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.     

Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.     

Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.     

Interaction of the essential Drosophila nuclear protein YA with P0/AP3 in the cytoplasm and in vitro: implications for developmental regulation of YA's subcellular location

The Drosophila nuclear lamina protein YA is essential for the transition from female meiosis to embryo mitosis. Its localization and, hence, function is under developmental and cell cycle controls. YA protein is hyperphosphorylated and cytoplasmic in ovaries. Upon egg activation, YA is partially dephosphorylated and acquires the ability to enter nuclei. Its function is first detected at this time. To investigate the cytoplasmic retention machinery that keeps YA from entering nuclei, we used affinity chromatography and blot overlay assays to identify cytoplasmic proteins that associate with YA. Drosophila P0/AP3, a ribosomal protein that is also an apurinic/apyrimidinic endonuclease, binds to YA in ovary and embryo cytoplasms. P0 and YA bind specifically and directly in vitro and are present in a 20S complex in the cytoplasmic extracts. YA protein can be phosphorylated by MAPK, but not by p34(Cdc2) kinase, in vitro. This phosphorylation increases YA's binding to P0. We propose that the P0-containing 20S cytoplasmic complex retains hyperphosphorylated ovarian YA in the cytoplasm. In response to egg activation, YA is partially dephosphorylated and its binding to the 20S complex is reduced. Hence, some YA dissociates from the complex and enters nuclei. Consistent with this model, decreasing P0 levels partially suppress a hypomorphic Ya mutant allele.     

The Drosophila maternal-effect gene fs(1)Ya encodes a cell cycle-dependent nuclear envelope component required for embryonic mitosis

The maternal-effect gene fs(1)Ya is specifically required for embryonic mitosis in Drosophila. fs(1)Ya is involved in the initiation of the first embryonic mitosis and may also be necessary for subsequent embryonic mitotic divisions. fs(1)Ya encodes a 91.3 kd hydrophilic protein containing two putative MPF phosphorylation target sites and two potential nuclear localization signals. This protein is synthesized during postoogenic maturation from its maternal RNA and persists throughout embryogenesis. In early embryos, the fs(1)Ya protein is localized to the nuclear envelope from interphase to metaphase. During anaphase and telophase, it is dispersed in the nucleoplasm and cytoplasm, a behavior that is different from that of both the nuclear envelope and lamins. These results suggest that the fs(1)Ya protein is a cell cycle-dependent component of the nuclear envelope that specifically functions in embryonic mitosis.     

Functional Dissection of YA, an Essential, Developmentally Regulated Nuclear Lamina Protein in Drosophila melanogaster

The Drosophila YA protein is a nuclear lamina component whose function is essential to initiate embryonic development. To identify regions of YA required for its action in its normal cellular context, we made targeted mutations in the YA protein and tested their consequences in flies and embryos in vivo. We found that critical amino acids are distributed along the length of the YA molecule, with functionally important regions including the N- and the C-terminal ends, the cysteine residues in YA’s two potential zinc fingers, a serine/threonine-rich region, and a potential maturation-promoting factor or mitogen-activated protein kinase phosphorylation target site, ITPIR. In addition, several Ya mutations showed intragenic complementation, with N-terminal mutations complementing C-terminal mutations, suggesting that YA proteins interact with one another. In support of this interaction, we demonstrated by immunoprecipitation that YA molecules are present in complexes with each other. Finally, we showed that the C-terminal 179 amino acids of YA are necessary to target, or retain, YA in the nuclear envelope.     

Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.     

Caenorhabditis elegans nucleoporins Nup93 and Nup205 determine the limit of nuclear pore complex size exclusion in vivo

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of approximately 70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.     

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.     

The Drosophila plutonium and pan gu genes regulate entry into S phase at fertilization

Mutations in the Drosophila maternal genes plutonium (plu) and pan gu (png) have the striking phenotype that DNA replication initiates in unfertilized eggs. Fertilized eggs from plu or png mutant mothers also have a mutant phenotype; DNA replication is uncoupled from nuclear division, resulting in giant, polyploid nuclei. Analysis of multiple alleles of these genes indicates that their wild-type function is required to maintain repression of DNA replication until fertilization. The phenotype of two png alleles suggests that this gene also may play a direct role in coupling S phase and mitosis during the early cleavage divisions. We describe genetic interactions among png, plu, and the previously identified gene gnu that demonstrate these three genes regulate the same process.     

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

A Highlights from MBoC Selection: A novel function for the Caenorhabditis elegans torsin OOC-5 in nucleoporin localization and nuclear import

Torsin proteins are AAA+ ATPases that localize to the endoplasmic reticular/nuclear envelope (ER/NE) lumen. A mutation that markedly impairs torsinA function causes the CNS disorder DYT1 dystonia. Abnormalities of NE membranes have been linked to torsinA loss of function and the pathogenesis of DYT1 dystonia, leading us to investigate the role of the Caenorhabditis elegans torsinA homologue OOC-5 at the NE. We report a novel role for torsin in nuclear pore biology. In ooc-5–mutant germ cell nuclei, nucleoporins (Nups) were mislocalized in large plaques beginning at meiotic entry and persisted throughout meiosis. Moreover, the KASH protein ZYG-12 was mislocalized in ooc-5 gonads. Nups were mislocalized in adult intestinal nuclei and in embryos from mutant mothers. EM analysis revealed vesicle-like structures in the perinuclear space of intestinal and germ cell nuclei, similar to defects reported in torsin-mutant flies and mice. Consistent with a functional disruption of Nups, ooc-5–mutant embryos displayed impaired nuclear import kinetics, although the nuclear pore-size exclusion barrier was maintained. Our data are the first to demonstrate a requirement for a torsin for normal Nup localization and function and suggest that these functions are likely conserved.     

Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes

We describe a stable cell-free mitotic extract derived from Xenopus eggs that contains activities necessary for nuclear envelope breakdown and chromosome condensation during mitosis. Using these cell-free extracts, we have demonstrated that nuclear envelope vesicularization, lamina solubilization, and chromosome condensation are independent and separable biochemical processes. We present evidence indicating that during mitosis nuclear membrane breakdown may involve the binding of a coating protein, lamin solubilization is enzymatically driven, and chromosome condensation involves both binding proteins and enzymatic activities including topoisomerase II. These results provide a coherent framework for investigating structural modification of the nucleus during mitosis at the biochemical level.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of embryogenesis.

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.