Sea urchin fertilization envelope: Uncoupling of cortical granule exocytosis from envelope assembly and isolation of an envelope intermediate from Strongylocentrotus purpuratus embryos

The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca²⁺- and Mg²⁺-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight.     

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.     

Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

Vitelline envelope gps 63 and 75 specifically bind sperm in "in vitro" assays in Discoglossus pictus

In Xenopus, conflicting data related to sperm-vitelline envelope (VE) binding suggest that further experiments should be performed to study the role of VE glycoproteins in sperm binding. In this article, we studied the VE of Discoglossus pictus, where gp63, the product of the Dp ZP2 gene, has high molecular identity to Xenopus gp69/64 and to mouse ZP2 and only A23187-treated sperm bind to VE. Sperm bind to VE all over the egg, yet a sperm tuft was found only in the animal half of the egg, where the dimple, the site of fertilization, is located and an intense immunostain was detected in VE by an antiserum directed against gp69/64. The same antiserum inhibited sperm binding to VE. Sperm binding to beads coated with gp63, gp40, or gp75 was in the range of 62-70% for gp63-beads, 67-75% for 75 beads, and about 20% for BSA beads and gp40-coated beads. Soluble purified gp63 and gp75 competitively inhibited binding of sperm to gp63-coated beads. Similarly, the same glycoproteins inhibited sperm binding to gp75-coated beads. SDS-polyacrylamide gels (PAGE) of FE and comparison of VE and FE peptide maps showed that gp63 undergoes a minor shift to about 62 kDa in FE. In sperm binding assays with beads coated with FEs gp62, there was no binding. Following fertilization, in the region of the dimple, an F-layer is formed as well as an alteration of the VE structure. Lectin blots of the FE showed that the FE and in particular gp62 acquires a stronger affinity to Maackia amurensis agglutinin (MAA) with respect to VEs gp63. These results indicate that gps 63 and 75 are the sperm binding glycoproteins of D. pictus VE, where major post-fertilization changes occur as in other anuran species.     

Physicochemical characterization of progressive changes in the Xenopus laevis egg envelope following oviductal transport and fertilization

Previous studies have shown that the Xenopus laevis egg envelope exists in three forms with differing ultrastructural, macromolecular, and sperm penetrability properties. The coelomic envelope (CE) is derived from eggs released from the ovary into the body cavity of the female, the vitelline envelope (VE) from eggs which have passed through the oviduct, and the fertilization envelope (FE) from fertilized eggs. In the present study, the physicochemical characteristics of these three envelope types were differentiated. Investigation of envelope solubility, deformability, sulfhydryl reactivity, and hydrophobic dye and ferritin binding capacity demonstrated that profound physicochemical changes occur in envelope conversions CE----VE----FE. The physical strength of the envelopes, as evidenced by deformability studies, ranked FE greater than CE greater than VE. These differences were not accountable by differences in the number of disulfide bonds, although the CE sulfhydryl groups were significantly less accessible than those in the VE or FE. All three envelope forms were hydrophilic in nature, exhibiting little ability to bind 1-anilino-8-naphthalenesulfonic acid. The CE bound greater amounts of ferritin in comparison to the VE and FE, indicating the presence of a basic domain, presumably in the 43-kDa glycoprotein, which is lost upon proteolysis to 41 kDa during the CE----VE conversion. The envelope integrity of all three forms was maintained by both noncovalent and covalent (disulfide) bonds. Measurements of the effect of pH on envelope solubilization indicated the involvement of an ionizable group with pKa of 8.0 in maintaining envelope structure.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro

The rainbow trout egg vitelline envelope (VE) is composed of three proteins, called VEalpha ( approximately 58-60kDa Mr), VEbeta ( approximately 52kDa Mr), and VEgamma ( approximately 47kDa Mr). Each of these proteins is related to mouse egg zona pellucida (ZP) glycoproteins, called ZP1, ZP2, and ZP3, and possesses a ZP domain that has been implicated in the polymerization of the proteins into long, interconnected fibrils or filaments. Here, trout egg VEbeta and VEgamma were purified to homogeneity and analyzed under various experimental conditions (SDS-PAGE, Blue Native-(BN-)PAGE, size-exclusion chromatography, and transmission electron microscopy) to determine whether individual VE proteins would polymerize into fibrils in vitro. Such analyses revealed that in the presence of 6M urea each VE protein is present primarily as monomers and as small oligomers (dimers, tetramers, etc.). However, either a reduction in urea concentration or a complete removal of urea results in the polymerization of VEbeta and VEgamma dimers into very large oligomers. Mixtures of VEbeta and VEgamma also give rise to large oligomers. Under these conditions, VE proteins are visualized by transmission electron microscopy as aggregates of long fibrils, with each fibril composed of contiguous beads located periodically along the fibril. The relationship between the behavior of fish egg VE proteins and mouse ZP glycoproteins, as well as other ZP domain-containing proteins, is discussed.     

Ultrastructure and Ultracytochemistry of Fertilization Envelope Formation in the Carp Egg

This paper describes ultrastructural and ultracytochemical events occurring during the process of transformation of the vitelline envelope (VE) into the fertilization envelope (FE) in the egg of Cyprinus carpio. The VE is composed of four layers, except the micropylar region. The outermost (first) layer can be subdivided into a double layer (upper and lower halves) by cytochemical differences. The upper half is more protein-rich and positive for acid phosphatase (AcPase) activity, while the lower one is more carbohydrate-rich and negative for AcPase.
The most striking differences between the VE and the FE appear in the first layer component and the thickness. The FE first layer, ultrastructurally and cytochemically consisting of a single layer, gradually grows to be about five-fold as thick as the VE first layer by 40 min after fertilization. This may be due to displacement of the former VE first layer by deposits of the cortical alveolar exudate.     

Enzymes responsible for the bactericidal effect in extracts of vitelline and fertilisation envelopes of rainbow trout eggs

Extracts from both the vitelline envelope (VE) and fertilisation envelopes (FE) of rainbow trout eggs have the ability to exert a bactericidal effect on Gram-positive and -negative bacteria. The effect may be due to the presence of phospholipase D (PLD), lysozyme, proteinase and DNases, as the extracts contain these enzyme activities. The intensity of chorionic PLD and lysozyme activities in the VE extract was maintained in the FE without any alteration in activity even after transformation in the course of the cortical reaction, as components of a fundamental architecture of the envelope. Both extracts also contain different types of proteinase activities. Treatment with VE or FE extract seriously damaged the outer membrane of Gram-negative bacteria and the plasma membrane of Gram-positive and -negative bacteria at the ultrastructural level. Chorionic DNases probably degrade DNA of bacterial cells killed by virtue of the action of PLD and/or lysozyme and contribute to the transmigration of nucleosides and/or nucleotides produced by degrading bacterial DNA after degradation of bacterial components by the actions of the chorionic PLD, lysozyme and proteinase. These results suggest that the bactericidal process manifested by the VE or FE extract may start with the action of PLD and/or lysozyme against bacteria and be completed by subsequent degradation of constitutive proteins and DNA by the action of proteinases and DNases, respectively. Thus the VE and FE are able to protect the egg itself and the embryo, respectively, from bacterial infection in the internal or external environments.     

The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FE_h) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one-dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FE_h were crossreactive with antibodies against Xenopus VE^*.     

Sulphated glycoconjugates are powerful inhibitors of spermatozoa binding to the vitelline envelope in amphibian eggs

BACKGROUND INFORMATION: In amphibians, the role of sulphated glycans has not been determined in spermatozoa-egg interaction, although they are known to be involved in other systems. In previous studies, it was found that, in Discoglossus pictus, a VE (vitelline envelope) glycoprotein of 63 kDa exhibits high homology to Xenopus laevis gp69/gp64 and to ZP2 of mammals. gp63 and a glycoprotein of 75 kDa are both capable of binding the spermatozoa in in vitro assays and, having similar peptide maps and different glycosylation, are probably two glycoforms of the same protein. RESULTS: In the present study, binding assays performed by treating dejellied eggs with metaperiodate suggest that hydroxy groups of sugars are not directly involved in spermatozoa-vitelline envelope binding. Competition assays between dejellied eggs and spermatozoa preincubated with dextran, dextran sulphate or fucoidan indicated that sulphated oligosaccharides have an inhibitory effect on spermatozoa binding. In similar competition assays, Le(x) (Lewis(x)) trisaccharide 3'-sulphate inhibited spermatozoa binding to VE in contrast with 3'-sialyl-Le(x) tetrasaccharide. Assays performed with gp75- or gp63-coated beads and spermatozoa treated with fucoidan or dextran sulphate indicated that sulphated oligosaccharides competitively inhibit spermatozoa binding to gp75-coated beads, yet not to gp63-coated beads. Finally, solubilized VE digested with N-glycosidase F retains the inhibitory activity in spermatozoa-VE binding assays in contrast with VE treated with alpha-N-acetylgalactosaminidase. CONCLUSION: It was concluded that VE sulphate groups are involved in spermatozoa binding. These groups are present in gp75 glycoconjugates and are probably located in O-linked glycoconjugates.     

Radioiodination studies of the envelopes from Xenopus laevis eggs

To investigate the molecular basis of the observed morphological and biological characteristics of coelomic egg envelopes (CE), vitelline envelopes (VE), and fertilization envelopes (FE) of Xenopus laevis eggs, envelopes were radioiodinated under a variety of conditions: in situ, isolated and intact, or solubilized. The distribution of 125I in envelope components was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each envelope type displayed unique profiles when iodinated in the intact state. A major constituent of VE, the 41,500 molecular weight component, was not labeled in the intact state, although the corresponding component of CE was heavily labeled. After dissociation of the envelope by guanidine-HCl or sodium dodecyl sulfate, all of the components could be radioiodinated. However, when the envelopes (VE and FE) were dissolved by heating and subsequently radioiodinated by lactoperoxidase, the resulting radioactivity profile was similar to that of the intact envelopes, suggesting that in the heat-dissolved envelope, the individual components retain similar structural relations as in the intact envelope. Quantitative but not qualitative differences were found between the inner and outer aspects of VE and FE. The significance of these findings is discussed in relation to what is known about the morphological, biological, and molecular properties of the envelopes.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Depletion of nucleoporins from HeLa nuclear pore complexes to facilitate the production of ghost pores for in vitro reconstitution

During cell division, Nuclear Pore Complexes (NPCs) are broken down into protein subcomplexes that are the basis for reassembly in daughter cells. This is the driving force for the establishment of an in vitro reconstitution system to study aspects of NPC reassembly. In this study, nuclear envelope (NE) was isolated from HeLa cells. NE was treated with increasing concentrations of heparin to extract nucleoporins (Nups) for the production of “ghost pores” which are pores severely deficient in Nups, while still containing Pore Membrane proteins (POM) needed to anchor the NPC. Ghost pores have been subjected to incubation with previously stripped Nups and some re-binding has been shown to occur by western blot analysis. This in vitro assay provides a powerful tool to investigate the protein–protein interactions of NPC reassembly from a human cell line. Through a better understanding of the process of NPC reassembly, we can continue to piece together the puzzle of this macromolecular structure. It is most advantageous to establish a straightforward reconstitution procedure at the mammalian level.     

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.     

Interaction between corneocytes and stratum corneum lipid liposomes in vitro

Small unilamellar vesicles were made from a mixture of epidermal ceramides (45%), cholesterol (35%), free fatty acids (15%) and cholesteryl sulfate (5%). Isolated corneocytes prepared from pig epidermis were added to the liposomes and the interaction between corneocytes and liposomes was studied by (1) thin-section electron microscopy and (2) monitoring the release of aqueous contents of the vesicles by following the fluorescence intensity of carboxyfluorescein entrapped in the vesicles. The vesicles adsorbed readily onto the corneocytes and slowly transformed into lamellar sheets. Enhanced fluorescence intensity indicated a corneocyte-induced membrane fusion process that resulted in the release of aqueous contents of the vesicles. The results suggest a cohesive role for the corneocyte cell envelope, which consists of a monomolecular layer of lipids covalently bound to the outside of a cross-linked protein envelope. This may be one of the major factors in the reassembly of extruded membranous disks into lamellar sheets which occurs during the final stages of epidermal differentiation.     

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.     

Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.     

A cytoskeletal 50 kDa protein in higher plants that forms intermediate-sized filaments and stabilizes microtubules

Summary Filamentous structures of 7–10 nm in diameter were regenerated in vitro from a soluble 50 kDa protein (p50) that had been isolated from mung bean seedlings and from cultured tobacco cells. The polymerization of p50 in vitro was dependent on the presence of guanosine nucleotides, in particular, guanosine monophosphate (GMP). Unlike tubulin, p50 is a stable basic protein with the ability to polymerize even after heat treatment for 1 min at 70 °C. Furthermore, the freeze-dried powder of p50 retained the ability to regenerate filamentous structures after it had been dissolved in polymerization buffer to which GMP was then added. Two monoclonal antibodies against p50 were obtained. These antibodies stained the filamentous structures that extended from the surface of the nucleus to the cell periphery in interphase tobacco cells. They stained spindles and phragmoplasts as did tubulin-specific antibodies. They also stained fibrillar structures that were present around the spindle poles and the telophase daughter nuclei in which no microtubules were present. These results suggest that a part of the cell's complement of p50 may be associated with microtubules in dividing cells while the rest may itself form unique fibrillar structures. The antibodies against p50 did not stain cortical microtubules or the pre-prophase band of microtubules. The antibody against p50 also stained intermediate filament-like structures in cultured animal cells. The formation of microtubules in vitro was markedly stimulated and the assembled microtubules were greatly stabilized by p50. Further investigation of p50 is indispensable for the understanding of properties and function of intermediate-sized filaments in higher plant cells.Abbreviations EPC Sepharose ethyl N-phenyl-carbamate conjugated Sepharose - p50 50 kDa protein