Dynamics of the annulate lamellae in Drosophila syncytial embryos

In eukaryotic cells, mitotic events are controlled by evolutionarily conserved cyclin-dependent kinases (cdk): these kinases phosphorylate cell proteins, which causes structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that cdk1 activity is a key factor that controls nuclear pore complex assembly/disassembly and affects the structure of cytoplasmic pores in the annulate. In this paper, we report a comparative analysis of these cytoplasmic organelles throughout the cell-cycle and throughout the development of Drosophila syncytial embryos. Based on the results obtained, it was presupposed that distribution of annulate lamellae containing cytoplasmic pores could reflect the inactivation of the mitotic kinase cdk1 in Drosophila syncytial embryos.     

Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components?

Annulate lamellae are cytoplasmic organelles composed of stacked sheets of membrane containing pores that are structurally indistinguishable from nuclear pores. The functions of annulate lamellae are not well understood. Although they may be found in virtually any eucaryotic cell, they occur most commonly in transformed and embryonic tissues. In Drosophila, annulate lamellae are found in the syncytial blastoderm embryo as it is cleaved to form the cellular blastoderm. The cytological events of the cellularization process are well documented, and may be used as temporal landmarks when studying changes in annulate lamellae. By using morphometric techniques to analyze electron micrographs of embryos, we are able to calculate the number of pores per nucleus in nuclear envelopes and annulate lamellae during progressive stages of cellularization. We find that annulate lamellae pores remain at a low level while nuclear envelopes are expanding and acquiring pores in early interphase. Once nuclear envelopes are saturated with pores, however, the number of annulate lamellae pores increases more than 10-fold in 9 min. Over the next 30 min it gradually declines to the initial low level. On the basis of these results, we propose (a) that pore synthesis and assembly continues after nuclear envelopes have been saturated with pores; (b) that these supernumerary pores accumulate transiently in cytoplasmic annulate lamellae; and (c) that because these pores are not needed by the embryo they are subsequently degraded.     

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.     

Monoclonal antibody raised against murine IL-1 α peptide cross-reacts with a 60-kDa antigen in early Drosophila melanogaster embryo

Whole-mounts of Drosophila embryos were stained with the monoclonal antibody Vmp 18, raised against the peptide 199–208 of murine interleukin 1/. 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.This work was supported in parts by grants from the Italian MURST (40% and 60% funds) and from the Consorzio Siena Ricerche     

Grapes(Chk1) prevents nuclear CDK1 activation by delaying cyclin B nuclear accumulation

Entry into mitosis is characterized by a dramatic remodeling of nuclear and cytoplasmic compartments. These changes are driven by cyclin-dependent kinase 1 (CDK1) activity, yet how cytoplasmic and nuclear CDK1 activities are coordinated is unclear. We injected cyclin B (CycB) into Drosophila melanogaster embryos during interphase of syncytial cycles and monitored effects on cytoplasmic and nuclear mitotic events. In untreated embryos or embryos arrested in interphase with a protein synthesis inhibitor, injection of CycB accelerates nuclear envelope breakdown and mitotic remodeling of the cytoskeleton. Upon activation of the Grapes(checkpoint kinase 1) (Grp(Chk1))-dependent S-phase checkpoint, increased levels of CycB drives cytoplasmic but not nuclear mitotic events. Grp(Chk1) prevents nuclear CDK1 activation by delaying CycB nuclear accumulation through Wee1-dependent and independent mechanisms.     

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling

Background

In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.

Results

Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof ¹ /+; mnk ^p6 /+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.

Conclusion

mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.     

Structural organization and possible functional role of annulate lamellae containing cytoplasmic pores

This review is devoted to annulate lamellae, a specific compartment of endoplasmic reticulum that occurs, presumably, in actively growing and rapidly dividing cells (oocytes, embryonic and tumor cells). We summarized both earlier and recent data on the dustribution of annulate lamellae in various cell types, on their morphology, and the distribution of interaction with intracellular structures at various treatments. As the annulate lamellae contain cytoplasmic pore complexes, a special attention was paid to their relation with nuclear pores. Possible functions of the annulate lamellae in intracellular processes and, particularly, in nuclear envelope assembly, are discussed.     

Insertional Mutation of the Drosophila Nuclear Lamin Dm0 Gene Results in Defective Nuclear Envelopes, Clustering of Nuclear Pore Complexes, and Accumulation of Annulate Lamellae

Nuclear lamins are thought to play an important role in disassembly and reassembly of the nucleus during mitosis. Here, we describe a Drosophila lamin Dm₀ mutant resulting from a P element insertion into the first intron of the Dm₀ gene. Homozygous mutant animals showed a severe phenotype including retardation in development, reduced viability, sterility, and impaired locomotion. Immunocytochemical and ultrastructural analysis revealed that reduced lamin Dm₀ expression caused an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. In several cells, particularly the densely packed somata of the central nervous system, defective nuclear envelopes were observed in addition. All aspects of the mutant phenotype were rescued upon P element-mediated germline transformation with a lamin Dm₀ transgene. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus.     

Fibrogranular Material,Annulate Lamellae and Microtubules During Spermiogenesis in Drosophila melanogaster

During spermiogenesis in Drosophila melanogaster, a “perinuclear plasm’ accumulates between the fenestrated portion of the nuclear envelope and an adjacent lamella of ER in the young spermatid. Microtubules appear within the perinuclear plasm and become especially concentrated in a nuclear concavity. Cytoplasmic pores are present locally within the lamella of ER. In addition, localized or discrete bodies composed of fibrogranular material become closely associated with single pore complexes in the lamella of ER. A close association exists between pore complexes (annulate lamellae), the small granular and fibrillar subunits of the fibrogranular bodies, polyribosomes and the nuclear-associated microtubules during much of spermiogenesis. While the fibrogranular material becomes less concentrated during spermiogenesis, the number of pore complexes in a single section increases such that two, three or even four short annulate lamellae are intercalated within many longitudinally oriented microtubules which are present in the furrow of the spermatid nucleus. Structural relationships observed between cytoplasmic pores (annulate lamellae), fibrogranular bodies, polyribosomes and microtubules are discussed in relation to information about the timing of RNA and protein synthesis. This study extends previous observations about the distribution and structural variations of annulate lamellae elsewhere in the spermatid cytoplasm.     

NEGATIVE STAINING AND ADENOSINE TRIPHOSPHATASE ACTIVITY OF ANNULATE LAMELLAE OF NEWT OOCYTES

Semi-isolated annulate lamellae were prepared from single newt oocytes (Triturus alpestris) by a modified Callan-Tomlin technique. Such preparations were examined with the electron microscope, and the negative staining appearance of the annulate lamellae is described. The annulate lamellae can be detected either adhering to the nuclear envelope or being detached from it. Sometimes they are observed to be connected with slender tubular-like structures interpreted as parts of the endoplasmic reticulum. The results obtained from negative staining are combined with those from sections. Especially, the structural data on the annulate lamellae and the nuclear envelope of the very same cell were compared. Evidence is presented that in the oocytes studied the two kinds of porous cisternae, namely annulate lamellae and nuclear envelope, are markedly distinguished in that the annulate lamellae exhibit a much higher pore frequency (generally about twice that found for the corresponding nuclear envelope) and have also a relative pore area occupying as much as 32% to 55% of the cisternal surface (compared with 13% to 22% in the nuclear envelopes). The pore diameter and all other ultrastructural details of the pore complexes, however, are equivalent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various animal and plant cells, the annuli of the annulate lamellae pores reveal also an eightfold symmetry of their subunits in negatively stained as well as in sectioned material. Furthermore, the annulate lamellae are shown to be a site of activity of the Mg-Na-K-stimulated ATPase.     

A multilamellate body in the granulated hypophysial cells of the teleost,Hemihaplochromis philander

Summary A multilamellate body (MLB), bearing close resemblance to an array of annulate lamellae, has been observed in several adenohypophysial cell types of the teleost, Hemihaplochromis philander. In longitudinal section, each MLB comprises a ladder-like row of 12–50 sausage-shaped profiles, termed lamellae. A few lamellae in each section show connections with the endoplasmic reticulum. Apposition of paired lamellar membranes at regularly spaced intervals results in a beaded appearance, whereby sites of membrane apposition are probable pore sites. The MLB differs from annulate lamellae in having poorly developed pores and closer packing of lamellae. It is suggested that the MLBs described here, may represent annulate lamellae at a stage of development or break-down when pores are incomplete.Acknowledgement is made to the South African Council for Scientific and Industrial Research for financial aid towards this work. The authors also wish to thank the Natal Parks, Game and Fish Preservation Board for providing specimens     

Karyomeres in early cleavage embryos of ophryotrocha labronica lagreca and bacci

Summary Karyomeres or chromosome vesicles occur regularly at all cell divisions in cleavage embryos ofOphryotrocha labronica up to the 16-cell stage. They are formed as separate units, containing one or several nucleolus-like bodies (NLB) as well as intranuclear annulate lamellae (IAL), but coalesce later into a compound nucleus, in connection with copious blebbing and simultaneous appearance of cytoplasmic annulate lamellae (CAL). Labelling of the early embryos with³H-thymidine revealed marked localization of the synthesized DNA to the karyomere envelope region, whereas³H-uridine incorporation, indicating RNA synthesis, was sparse and notably absent in the NLB. On the other hand the latter structure like the envelopes preferentially incorporated³H-myoinositol, and displayed considerable labelling with³H-leucine. The mechanism and general significance of karyomere formation is discussed with particular attention to the NLB and their possible involvement in nuclear membrane formation.This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.The excellent technical assistance of Mrs Annagreta Petersen and Mrs Lena Olsson is gratefully acknowledged.     

Annulate lamellae and "yolk nuclei" in oocytes of the dragonfly, Libellula pulchella

Annulated membranes in the form of single and short lamellae are present adjacent to and parallel to the nuclear envelope in oogonia and early oocyte (synaptene) stages of the dragonfly, Libellula pulchella. These solitary and short annulate lamellae are usually continuous with long, part rough- and part smooth-surfaced cisternae which extend into more distal areas of the oogonial ooplasm. These particular annulate lamellae then either disappear or decrease in number to be replaced by a much more extensive system of annulate lamellae in the cortical ooplasm of previtellogenic oocytes. The differentiation of extensive stacks of annulate lamellae is consistently observed to be restricted to large cytoplasmic areas of considerable electron density. These cytoplasmic regions consist of material which stains basophilic and contains RNA but differs structurally from the large number of ribosomes which surround the dense masses. The cytoplasmic dense masses, in terms of their formation and staining reactions, are comparable to the "yolk nuclei" or "Balbiani bodies" described in insect oocytes in earlier studies. The results of the present study thus provide evidence that the appearance of cortical ooplasmic stacks of annulate lamellae in the dragonfly oocyte is specifically limited to cytoplasmic areas of high electron density which contain RNA but which do not have a ribosomal morphology.     

Live imaging reveals that the Drosophila actin-binding ERM protein, moesin, co-localizes with the mitotic spindle

Members of the vertebrate ezrin-radixin-moesin (ERM) protein family crosslink the actin cytoskeleton and the cell membrane and are, therefore, considered cytoplasmic regulators of cell adhesion, cell movement and membrane trafficking. Here we demonstrate that besides its cytoplasmic functions Drosophila moesin, the only ERM protein in Drosophila melanogaster, exhibits a dynamic cell cycle-dependent nuclear localization. In a small fraction of cells and at a low level, moesin can be detected in interphase nuclei in regions complementary to the chromatin; its level rapidly increases during prophase and it co-localizes with the actin network surrounding the mitotic spindles throughout mitosis. We also found that the predicted single nuclear localization signal in moesin is not necessary for the nuclear accumulation of the protein. FRAP experiments confirmed this finding and further revealed that the mitotic localization of moesin is highly dynamic. Immuno-histochemical staining for moesin demonstrated the existence of spindle association in wild-type embryos. The biological relevance of this phenomenon is indicated by the mitotic phenotypes detected in S2 cells treated with moesin RNAi, and awaits future exploration.     

Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

A PECULIAR CONFIGURATION OF AGRANULAR RETICULUM (CANALICULATE LAMELLAR BODY) IN THE RAT PINEALOCYTE

A cytoplasmic structure exhibiting a peculiar configuration of agranular reticulum has been found in the rat pinealocyte, and has been designated a canaliculate lamellar body. It consists of a number of fenestrated, flat cisternae which are closely spaced. They bear some distant resemblance to the annulate lamellae previously reported in a variety of cell types. Profiles of stacks of lamellae in a plane of section always display two distinct aspects, the surface and the cross-sectional views of flat cisternae. A surface view shows the hexagonal arrangement of pores or fenestrations. The pores in successive lamellae are aligned precisely, one behind the other, so that clear, cylindrical channels are seen running perpendicular to the lamellae as indicated by transverse sections of the lamellar stacks. Large canaliculate lamellar bodies are composed of many extended series of lamellar stacks which pursue a tortuous course and cross one another. Occasionally the canaliculate lamellar body is located deep in a nuclear invagination, which reminds one of the so-called nuclear pellets (Kernkugeln) reported by light microscopy. The functional significance of the body is unknown.     

Cytoplasmic syncytial interneuronal connection in molluscs

The absolute criteria developed by the authors have been presented; they allow revealing cytoplasmic syncytial connections between processes of nerve cells in vivo and in vitro at the light microscopy level by using classical methods and time lapse videoshooting in the phase contrast. With aid of electron microscopy, metastable membrane contacts and their perforations, cytoplasmic syncytial interneuronal pores, and fusion of nerve processes are demonstrated. In the culture of isolated molluscan neurons, the process of formation of syncytial connections between processes of the same neuron or of different neurons is reproduced. Processes of one neuron, which have syncytial connection with another neuron, are shown to remain viable after death of its neuronal soma. The cytoplasmic varicosities formed on processes of one neuron are able to overcome the place of syncytial contact with processes of another neuron and to move to the body of the latter. A hypothesis is put forward that the cytoplasmic syncytial connection between nerve processes is formed under the conditions of the absence of their glial sheaths.     

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis

Summary During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 m from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.