Microtubule-dependent assembly of the nuclear envelope in Xenopus laevis egg extract

Microtubules take part in several mechanisms of intracellular motility, including organelle transport and mitosis. We have studied the ability of Xenopus egg extract to support nuclear membrane and pore complex formation when microtubule dynamics are manipulated. In this report we show that the formation of a nuclear envelope surrounding sperm chromatin requires polymerized microtubules. We have observed that microtubule-depolymerizing reagents, and AS-2, a known inhibitor of the microtubule motor protein kinesin, do not inhibit the formation of a double nuclear membrane. However these double membranes contain no morphologically identifiable nuclear pore complexes and do not support the accumulation of karyophilic proteins. In contrast, the assembly of annulate lamellae, cytoplasmic structures containing a subset of pore complex proteins, was not affected. Our data show that not only polymerized microtubules, but also the microtubule motor protein kinesin, are involved in the formation of the nuclear envelope. These results support the conclusion that multiple nuclear envelope-forming mitotic vesicle populations exist, that microtubules play an essential and selective role in the transport of nuclear envelope-forming vesicle population(s), and that separate mechanisms are involved in nuclear envelope and annulate lamellae formation.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Chromatin assembly in Xenopus oocytes: In vitro studies

We describe and characterize a complex reaction that catalyzes DNA supercoiling and chromatin assembly in vitro. A Xenopus oocyte extract supplemented with ATP and Mg⁺⁺ converts DNA circles into minichromosomes that display a native, 200 bp periodicity. When supercoiled DNA is added to this extract it undergoes a time-dependent series of topological changes, which precisely mimic those found when the DNA is microinjected into oocytes. As judged by the conformation of the subsequently deproteinized DNA, the supercoiled DNA is first relaxed, in a reaction that takes 4 min, and then it is resupercoiled in a slower process that takes 4 hr. The relaxation is partially inhibited by EDTA, to an extent that suggests that that it is catalyzed by a type I DNA topoisomerase. The resupercoiling, on the other hand, requires ATP and Mg⁺⁺, is completely inhibited by EDTA, and is inhibited by novobiocin in a manner that suggests it is catalyzed by a type II DNA topoisomerase. These findings, and the ones reported in the preceding paper (Ryoji and Worcel, 1984), lead us to propose that chromatin assembly is an active, ATP-driven process.     

Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts

Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes approximately 1.5 h to prepare the extract, 1-3 h for spindle-assembly experiments and an additional 1-3 h if immunodepletion is performed.     

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.     

Microtubule plus-end dynamics in Xenopus egg extract spindles

Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at approximately 11 microm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization ( approximately 30 microm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by alpha-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.     

In vitro activation of human sperm induced by amphibian egg extract

In this paper we characterize a system for the activation of human sperm using cell-free extracts from Xenopus laevis eggs. We characterize the kinetics of sperm activation in terms of morphological and DNA synthetic parameters. We have optimized some of the components which are necessary for chromatin decondensation and present data to demonstrate that the in vitro process is efficient over a wide range of salt concentrations and amounts of supplementary reducing agents.     

pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract

In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.     

Nucleologenesis: Composition and fate of prenucleolar bodies

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently reorganize and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.     

Functional analysis of Survivin in spindle assembly in Xenopus egg extracts

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.     

Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.     

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.     

In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.     

CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least approximately 2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.     

Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 μm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.     

Bipolarization and poleward flux correlate during Xenopus extract spindle assembly

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.