Cell-free Extracts Of Xenopus Eggs Research Articles

DNA Content Contributes to Nuclear Size Control in <i>Xenopus</i>

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size, remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects as constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found an increase in the amount of membranes on the nuclear surface when DNA content was low and alterations in the nuclear expansion dynamics after converting the chromatin structures. These results demonstrate a novel model in which the physical properties of the chromatin, rather than the coding sequences themselves, contribute to nuclear expansion by generating forces that expand the nucleus outwardly.

Light-inducible activation of cell cycle progression in Xenopus egg extracts under microfluidic confinement.

Cell-free Xenopus egg extract is a widely used and biochemically tractable model system that allows recapitulation and elucidation of fundamental cellular processes. Recently, the introduction of microfluidic extract manipulation has enabled compartmentalization of bulk extract and a newfound ability to study organelles on length scales that recapitulate key features of cellular morphology. While the microfluidic confinement of extracts has produced a compelling platform for the in vitro study of cell processes at physiologically-relevant length scales, it also imposes experimental limitations by restricting dynamic control over extract properties. Here, we introduce photodegradable polyethylene glycol (PEG) hydrogels as a vehicle to passively and selectively manipulate extract composition through the release of proteins encapsulated within the hydrogel matrix. Photopatterned PEG hydrogels, passive to both extract and encapsulated proteins, serve as protein depots within microfluidic channels, which are subsequently flooded with extract. Illumination by ultraviolet light (UV) degrades the hydrogel structures and releases encapsulated protein. We show that an engineered fluorescent protein with a nuclear localization signal (GST-GFP-NLS) retains its ability to localize within nearby nuclei following UV-induced release from hydrogel structures. When diffusion is considered, the kinetics of nuclear accumulation are similar to those in experiments utilizing conventional, bulk fluid handling. Similarly, the release of recombinant cyclin B Δ90, a mutant form of the master cell cycle regulator cyclin B which lacks the canonical destruction box, was able to induce the expected cell cycle transition from interphase to mitosis. This transition was confirmed by the observation of nuclear envelope breakdown (NEBD), a phenomenological hallmark of mitosis, and the induction of mitosis-specific biochemical markers. This approach to extract manipulation presents a versatile and customizable route to regulating the spatial and temporal dynamics of cellular events in microfluidically confined cell-free extracts.

Cell-free Xenopus egg extracts for studying DNA damage response pathways.

In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review, we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol ĸ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts.

Poly(ADP-ribose)-binding promotes Exo1 damage recruitment and suppresses its nuclease activities

Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5′ flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.

Reconstitution of cortical actin networks within water-in-oil emulsions.

We describe the reconstitution of dynamic cortical actin networks within cell-like compartments. The approach is based on encapsulation of Xenopus egg extract within water-in-oil emulsions. The growth of cortical actin networks is catalyzed by an amphiphilic actin nucleation-promoting factor that localizes to the water/oil interface. We first describe the preparation of cell-free Xenopus egg extract that provides all the soluble components of the actin machinery. We then describe the preparation of the amphiphilic, fluorescent actin nucleation-promoting factor that directs actin polymerization to the interface. Finally, we describe the steps required for assembly of dynamic actin cortices within water-in-oil emulsions, including the emulsification process and the sample preparation procedures. We provide recommendations for handling sensitive system components and discuss potential uses of this reconstitution approach for cytoskeletal research.

Developmental regulation of histone H2A deposition via serine-1 phosphorylation

H2A/H4 serine 1 phosphoryation (S1ph) is hypothesized to participate in many aspects of chromatin metabolism, including DNA replication, repair, recombination, mitosis, and regulation of gene expression. However, few specific functions for this ubiquitous histone modification have been demonstrated. In early Xenopus laevis embryos, development is tightly regulated to prevent premature zygotic transcription post-fertilization and to activate zygotic gene expression in a timely manner simultaneous with the mid-blastula transition (MBT). Rapid histone deposition occurs in Xenopus embryos and in sperm pronuclei assembled in cell-free Xenopus egg extract, concomitant with sperm decondensation and DNA replication. In vivo we observed that H2AS1ph appeared upon oocyte germinal vesicle breakdown (GVBD) while H4S1ph only appeared post-MBT. This is the first study to demonstrate a biological distinction between H2A and H4 S1 phosphorylation. H2A S1ph abundance in chromatin was not dependent upon DNA replication nor did it exhibit any cell-cycle changes in chromatin in cycling cell-free extract. However H4S1ph was observed on pronuclei assembled in cell-free extract in a DNA concentration-dependent fashion. The appearance of H4S1P highly correlates with the increased nuclear-to-cytoplasmic ratio found at the beginning of maternal gene expression at the MBT. Addition of wildtype H2A (H2Awt), H2AS1A (unphosphorylatable), and H2AS1E (phosphomimetic) mutant histone H2A/H2B dimers to cell-free extract showed that H2AS1A had a slower rate of chromatin incorporation than either H2Awt or H2AS1E. In cell-free extract H2Awt and H2AS1E, but not the unphosphorylated H2AS1A, interact with the histone storage chaperone Nucleoplasmin. All three interact with Nap1. Finally, overexpression of H2AS1A in Xenopusembryos results in more than 50% embryonic lethality. We conclude that H2A serine 1 phosphorylation is necessary for proper entrance into the histone chaperone pathway in the early embryo.

The Xenopus Cell Cycle: An Overview

Oocytes, eggs and embryos from the frog Xenopus laevis have been an important model system for studying cell-cycle regulation for several decades. First, progression through meiosis in the oocyte has been extensively investigated. Oocyte maturation has been shown to involve complex networks of signal transduction pathways, culminating in the cyclic activation and inactivation of Maturation Promoting Factor (MPF), composed of cyclin B and cdc2. After fertilisation, the early embryo undergoes rapid simplified cell cycles which have been recapitulated in cell-free extracts of Xenopus eggs. Experimental manipulation of these extracts has given a wealth of biochemical information about the cell cycle, particularly concerning DNA replication and mitosis. Finally, cells of older embryos adopt a more somatic-type cell cycle and have been used to study the balance between cell cycle and differentiation during development.

Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro–formed nuclei for Western blotting and scanning electron microscopy (SEM)

This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2-3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.

Nuclear Localization Signal Peptides Induce Molecular Delivery along Microtubules

Many essential processes in eukaryotic cells depend on regulated molecular exchange between its two major compartments, the cytoplasm and the nucleus. In general, nuclear import of macromolecular complexes is dependent on specific peptide signals and their recognition by receptors that mediate translocation through the nuclear pores. Here we address the question of how protein products bearing such nuclear localization signals arrive at the nuclear membrane before import, i.e., by simple diffusion or perhaps with assistance of cytoskeletal elements or cytoskeleton-associated motor proteins. Using direct single-particle tracking and detailed statistical analysis, we show that the presence of nuclear localization signals invokes active transport along microtubules in a cell-free Xenopus egg extract. Chemical and antibody inhibition of minus-end directed cytoplasmic dynein blocks this active movement. In the intact cell, where microtubules project radially from the centrosome, such an interaction would effectively deliver nuclear-targeted cargo to the nuclear envelope in preparation for import.

The APC Tumor Suppressor Promotes Transcription-Independent Apoptosis In vitro

The APC tumor suppressor is found in nonproliferating epithelial cells of the colonic crypts and is mutated in most colorectal tumors. To understand the function of APC in normal epithelium and how its loss leads to tumor formation, we tested whether APC is a mediator of apoptosis using an in vitro assay that monitors caspase-3-mediated cleavage of lamin B protein or a colorimetric substrate in a cell-free Xenopus egg extract. Recombinant APC protein accelerates apoptosis-associated caspase activity independently of ongoing transcription and protein synthesis. Conversely, the addition of mutant APC and immunodepletion of Xenopus APC decelerates apoptosis-associated caspase activity. Acceleration of apoptosis by APC is abolished by the caspase-8 inhibitor Z-IETD-FMK, demonstrating that caspase-8 is an essential component of APC-mediated apoptosis. These results suggest that the induction of apoptosis may be one role of APC in tumor suppression and that this mechanism is independent of beta-catenin-mediated effects on transcription.

Potential roles of the nucleotide exchange factor ECT2 and Cdc42 GTPase in spindle assembly in Xenopus egg cell-free extracts.

The ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho family of small GTPases. ECT2 contains motifs of cell cycle regulators at its N-terminal domain. We previously showed that ECT2 plays a critical role in cytokinesis. Here, we report a potential role of XECT2, the Xenopus homologue of the human ECT2, in spindle assembly in cell-free Xenopus egg extracts. Cloned XECT2 cDNA encodes a 100 kDa protein closely related to human ECT2. XECT2 is specifically phosphorylated in M phase extracts. Affinity-purified anti-XECT2 antibody strongly inhibited mitosis in Xenopus cell-free extracts. Instead of bipolar spindles, where chromosomes are aligned at the metaphase plane in control extracts, the addition of anti-XECT2 resulted in the appearance of abnormal spindles including monopolar and multipolar spindles as well as bipolar spindles with misaligned chromosomes. In these in vitro synthesized spindle structures, XECT2 was found to tightly associate with mitotic spindles. The N-terminal half of XECT2 lacking the catalytic domain also strongly inhibited spindle assembly in vitro, resulting in the formation of mitotic spindles with a low density. Among the representative Rho GTPases, a dominant-negative form of Cdc42 strongly inhibited spindle assembly in vitro. These results suggest that the Rho family GTPase Cdc42 and its exchange factor XECT2 are critical regulators of spindle assembly in Xenopus egg extracts.

The interaction of two classes of nuclear vesicles is induced by the dissociation of soluble proteins from one class of vesicles

The regulatory mechanism of fusion steps in nuclear envelope assembly was studied in vitro using cell-free extracts of Xenopus eggs. The nuclear envelope is formed only around the chromatin at the end of mitosis. Two kinds of vesicles are required for nuclear envelope assembly (J. Cell Biol. 112 (1991) 545). Light vesicles (LVs) have the chromatin-binding activity, but cannot fuse solely. On the other hand, heavy vesicles (HVs) neither bind to the chromatin nor LVs, but are needed for the fusion event. Therefore, the association of HVs with LVs that bind to the chromatin is required at a first step during the fusion of vesicles. We found that salt-treated HVs inhibited the binding of LVs to the chromatin. In addition, when salt-treated HVs were pretreated with the proteins in the residue of salt-treatment of HVs, LVs recovered the chromatin-binding activity. In contrast, salt-treatment of LVs did not influence the binding of LVs with chromatin. By using a fluorescence microscopy assay, we showed directly that salt-treated HVs associated with LVs or salt-treated LVs, but HVs did not. These results suggest that soluble mask proteins on HVs keep the two distinct vesicles separate. We propose that the dissociation of mask protein on HVs allows HVs to bind with LVs that are located on the chromatin, subsequently the fusion of each vesicle occurs and the nuclear envelope is formed.

Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP 3-dependent mechanism

Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase Cγ (PLCγ), and inositol-1,4,5-trisphosphate (IP 3)-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H 2O 2), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLCγ, and IP 3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP 3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H 2O 2 action in the extracts might involve tyrosine phosphorylation and activation of PLCγ, accompanied by the increase in IP 3 content and subsequent calcium release from IP 3-regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction.

Control of chromosomal DNA replication in the early Xenopus embryo.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Reconstitution of sperm nuclei of zebrafish (Danio rerio) in Xenopus egg extracts.

Cell-free extracts of Xenopus eggs cause cyclic change in permeabilized sperm nucleus, nuclear envelope breakdown, chromosome condensation, and reformation of nuclei. In this study, the ability of cell-free extracts to cause similar changes in zebrafish sperm was examined. When lysolecithin-treated sperm from zebrafish were incubated in Xenopus egg extracts, a series of changes in sperm nuclear morphology were observed periodically. These changes correlated with maturation-promoting factor (MPF) activity. Furthermore, sperm nuclei of zebrafish replicated DNA during reconstitution in Xenopus egg extracts. These results showed that cell-free extracts of Xenopus egg possess the ability to cause cell-cycle-dependent changes in zebrafish sperm, implying the possibility of generating transgenic zebrafish in a similar way to transgenic Xenopus.

Incorporation of the nuclear pore basket protein nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell-free extracts of Xenopus eggs.

In cell-free extracts of Xenopus eggs that support the assembly of replication-competent nuclei, we found that lamin B(3) specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif-containing nucleoporins. In vitro translated Nup153 and lamin B(3) co-immunoprecipitate, and lamin B(3) interacts specifically with the C-terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG-containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant-negative mutant XlaminB delta 2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG-containing nucleoporins and Nup93 are recruited normally. When the lamina of pre-assembled nuclei is disrupted using the same dominant-negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

Xenopus Cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G(1) and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends.

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

INDUCTION OF DNA SYNTHESIS OR APOPTOSIS IN MAMMALIAN NUCLEI BY XENOPUS EGG EXTRACTS THAT FAIL TO SUPPORT THE REPLICATION OF SPERM CHROMATIN

Cell-free extracts of Xenopus eggs are widely used to study DNA replication in higher eukaryotes. However, with Xenopus sperm chromatin as template, it is a common experience that the efficiency of replication varies from extract to extract, for reasons that are unclear. Here we show that the majority of extracts unable to support sperm replication are nevertheless able to induce DNA synthesis in intact mammalian nuclei. Those that do not, induce apoptosis.

Reaper-induced dissociation of a Scythe-sequestered cytochrome c-releasing activity.

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. We recently reported that Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. We now show that Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper which is detectably homologous to Grim and Hid is dispensable for Scythe binding.