Xenopus egg extract
Xenopus egg extract is a lysate that is prepared by crushing the eggs of the African clawed frog Xenopus laevis. It offers a powerful cell-free (or in vitro) system for studying various cell biological processes, including cell cycle progression, nuclear transport, DNA replication and chromosome segregation. It is also called Xenopus egg cell-free system or Xenopus egg cell-free extract.
History
[edit]The first frog egg extract was reported in 1983 by Lohka and Masui.[1] This pioneering work used eggs of the Northern leopard frog Rana pipiens to prepare an extract. Later, the same procedure was applied to eggs of Xenopus laevis, becoming popular for studying cell cycle progression and cell cycle-dependent cellular events.[2] Extracts derived from eggs of the Japanese common toad Bufo japonicus[3] or of the Western clawed frog Xenopus tropicalis[4] have also been reported.
Basics of extract preparation
[edit]The cell cycle of unfertilized eggs of X. laevis is arrested highly synchronously at metaphase of meiosis II. Upon fertilization, the metaphase arrest is released by the action of Ca2+ ions released from the endoplasmic reticulum, thereby initiating early embryonic cell cycles that alternates S phase (DNA replication) and M phase (mitosis).[5]
M phase extract
[edit]
Unfertilized eggs in a buffer containing the Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) are packed into a centrifuge tube. After removing excess buffer, the eggs are crushed by centrifugation (~10,000 g). A soluble fraction that appears between the lipid cap and the yolk is called an M phase extract. This extract contains a high level of cyclin B-Cdk1. When demembranated sperm nuclei are incubated with this extract, it undergoes a series of structural changes and is eventually converted into a set of M phase chromosomes with bipolar spindles.
S phase (interphase) extract
[edit]When CaCl₂ is added to an M-phase Xenopus egg extract at a concentration sufficient to override residual EGTA (typically several hundred micromolar), it triggers rapid inactivation of maturation promoting factor (MPF). This occurs through cyclin B degradation by the proteasome and induces cell cycle progression from metaphase to anaphase, and eventually into S phase. The resulting extract is referred to as an S-phase extract or interphase extract. Upon the addition of sperm chromatin to an S-phase extract, nuclear assembly is initiated: membrane vesicles accumulate around decondensed chromatin, fuse to form a nuclear envelope, and produce fully functional nuclei. Active nuclear transport occurs across the nuclear envelope, and DNA replication is initiated within the reconstituted nuclei. Because these extracts contain abundant mRNA and ribosomes, protein translation also takes place. Thus, this cell-free system can faithfully recapitulate many cellular events characteristic of proliferating cells. A notable exception is transcription, which does not occur in this system. This reflects the natural state of the Xenopus egg and early embryo, where transcription is largely repressed from the meiotic stages through to the blastula stage after fertilization.[6]
Different types of egg extract
[edit]Cycling extract
[edit]
Cycling extract is a cell-free system derived from Xenopus eggs that autonomously undergoes repeated S phase and M phase cycles. It is prepared by artificially inducing intracellular responses that mimic fertilization, typically through calcium ionophore treatment or electrical stimulation of unfertilized eggs, followed by mechanical crushing of the eggs.[7] This extract has been instrumental in elucidating fundamental mechanisms of cell cycle regulation. For example, it revealed that entry into M phase depends on the translation of cyclin B, which in turn activates maturation promoting factor (MPF).[8] Cycling extracts are primarily used to study the regulation of cell cycle progression.
High-speed supernatant (HSS)
[edit]High-speed supernatant (HSS) is a fraction obtained by ultracentrifuging a conventional Xenopus egg extract at 100,000–200,000 × g, which removes membrane components and ribosomes, leaving a solution enriched in soluble proteins. Although HSS lacks the capacity to support nuclear assembly or protein translation, it can partially recapitulate chromatin structural changes in a cell cycle–dependent manner.[2] It is particularly suitable for protein purification.
Nucleoplasmic extract (NPE)
[edit]Nucleoplasmic extract (NPE) is prepared from Xenopus egg extracts by first assembling nuclei in S-phase extract through the addition of a high concentration of sperm chromatin (~10,000 nuclei per μL). The reaction mixture is then centrifuged without dilution to separate the nuclei, which form a distinct layer at the top. This nuclear fraction is collected and further centrifuged at high speed, yielding a soluble supernatant (nucleoplasm) and a pellet containing nuclear membranes and chromatin. The supernatant is referred to as the nucleoplasmic extract (NPE). When DNA is pre-incubated in S-phase HSS and then NPE is added, DNA replication can be initiated without the need for nuclear envelope formation—a significant distinction from standard S-phase extract protocols, where replication initiation requires nuclear assembly.[9] This system has enabled high-resolution analysis of replication initiation mechanisms. Moreover, NPE supports efficient replication of plasmid DNA and other non-sperm-derived templates. Leveraging this property, researchers have also used NPE to investigate DNA repair pathways using exogenously damaged DNA substrates.[10]
Discoveries made using egg extracts
[edit]- Purification of M-phase promoting factor (MPF)[11]
- Elucidation of the role of synthesis and degradation of cyclin B in cell cycle progression[8][12]
- Discovery that degradation of a protein(s) other than cyclin B is necessary for initiating chromosome segregation[13]
- Discovery of a mechanism of spindle assembly that depends on chromatin, but not centrosomes[14]
- Proposal of a DNA replication licensing system[15] and identification of its responsible factor[16]
- Identification of importin α/β responsible for nuclear transport[17]
- Discovery of the condensin complex essential for mitotic chromosome assembly[18][19]
- Identification of the cohesin complex essential for sister chromatid cohesion[20]
More recently, the egg extracts have been used to study reprogramming of differentiated nuclei,[21] physical properties of spindles[22] and nuclei,[23] and theoretical understanding of cell cycle control.[24]
See also
[edit]- Yoshio Masui
- cell cycle
- Cdk1
- cyclin
- DNA replication
- nuclear transport
- spindle apparatus
- condensin
- cohesin
References
[edit]- ^ Lohka MJ, Masui Y (1983). "Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components". Science. 220 (4598): 719–721. Bibcode:1983Sci...220..719L. doi:10.1126/science.6601299. PMID 6601299.
- ^ a b Lohka MJ, Maller JL. (1985). "Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts". J. Cell Biol. 101 (2): 518–523. doi:10.1083/jcb.101.2.518. PMC 2113692. PMID 3926780.
- ^ Ohsumi K, Katagiri C (1991). "Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin". Dev. Biol. 148 (1): 295–305. doi:10.1016/0012-1606(91)90338-4. PMID 1936566.
- ^ Brown KS, Blower MD, Maresca TJ, Grammer TC, Harland RM, Heald R (2007). "Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle". J. Cell Biol. 176 (6): 765–770. doi:10.1083/jcb.200610043. PMC 2064050. PMID 17339377.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Masui Y (2000). "The elusive cytostatic factor in the animal egg". Nat. Rev. Mol. Cell Biol. 1 (3): 228–232. doi:10.1038/35043096. PMID 11252899. S2CID 5303121.
- ^ Newport J, Kirschner M (1982). "A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage". Cell. 30 (3): 675–686. doi:10.1016/0092-8674(82)90272-0. PMID 6183003.
- ^ Murray AW (1991). "Cell cycle extracts". Methods Cell Biol. 36: 581–605. doi:10.1016/S0091-679X(08)60298-8. PMID 1839804.
- ^ a b Murray AW, Kirschner MW (1989). "Cyclin synthesis drives the early embryonic cell cycle". Nature. 339 (6222): 275–280. Bibcode:1989Natur.339..275M. doi:10.1038/339275a0. PMID 2566917.
- ^ Walter J, Sun L, Newport J (1998). "Regulated chromosomal DNA replication in the absence of a nucleus". Mol. Cell. 1 (4): 519–529. doi:10.1016/S1097-2765(00)80052-0. PMID 9660936.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Räschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Schärer OD, Walter JC (2008). "Mechanism of replication-coupled DNA interstrand crosslink repair". Cell. 134 (6): 969–980. doi:10.1016/j.cell.2008.08.030. PMID 18805090.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Lohka MJ, Hayes MK, Maller JL (1988). "Purification of maturation-promoting factor, an intracellular regulator of early mitotic events". Proc. Natl. Acad. Sci. USA. 85 (9): 3009–3013. Bibcode:1988PNAS...85.3009L. doi:10.1073/pnas.85.9.3009. PMC 280132. PMID 3283736.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Murray AW, Solomon MJ, Kirschner MW (1989). "The role of cyclin synthesis and degradation in the control of maturation promoting factor activity". Nature. 339 (6222): 280–286. Bibcode:1989Natur.339..280M. doi:10.1038/339280a0. PMID 2566918. S2CID 4319201.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Holloway SL, Glotzer M, King RW, Murray AW (1993). "Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor". Cell. 73 (7): 1393–1402. doi:10.1016/0092-8674(93)90364-v. PMID 8391932. S2CID 26338475.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Heald R, Tournebize R, Blank T, Sandaltzopoulos R, Becker P, Hyman A, Karsenti E (1996). "Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts". Nature. 382 (6590): 420–425. Bibcode:1996Natur.382..420H. doi:10.1038/382420a0. PMID 8684481. S2CID 4238425.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Blow JJ, Laskey RA (1988). "A role for the nuclear envelope in controlling DNA replication within the cell cycle". Nature. 332 (6164): 546–548. Bibcode:1988Natur.332..546B. doi:10.1038/332546a0. PMID 3357511. S2CID 4313693.
- ^ Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H (1995). "Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor". Cell. 81 (4): 601–609. doi:10.1016/0092-8674(95)90081-0. PMID 7758114. S2CID 18797719.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Görlich D, Prehn S, Laskey RA, Hartmann E (1994). "Isolation of a protein that is essential for the first step of nuclear protein import". Cell. 79 (5): 767–778. doi:10.1016/0092-8674(94)90067-1. PMID 8001116. S2CID 7539929.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Hirano T, Mitchison TJ (1994). "A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro". Cell. 79 (3): 449–458. doi:10.1016/0092-8674(94)90254-2. PMID 7954811. S2CID 24140495.
- ^ Hirano T, Kobayashi R, Hirano M (1997). "Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein". Cell. 89 (4): 511–521. doi:10.1016/s0092-8674(00)80233-0. PMID 9160743. S2CID 15061740.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Losada A, Hirano M, Hirano T (1998). "Identification of Xenopus SMC protein complexes required for sister chromatid cohesion". Genes Dev. 12 (13): 1986–1997. doi:10.1101/gad.12.13.1986. PMC 316973. PMID 9649503.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Ganier O, Bocquet S, Peiffer I, Brochard V, Arnaud P, Puy A, Jouneau A, Feil R, Renard JP, Méchali M (2011). "Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors". Proc Natl Acad Sci USA. 108 (42): 17331–17336. doi:10.1073/pnas.1100733108. PMC 3198361. PMID 21908712.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Shimamoto Y, Maeda YT, Ishiwata S, Libchaber AJ, Kapoor TM (2011). "Insights into the micromechanical properties of the metaphase spindle". Cell. 145 (7): 767–778. doi:10.1016/j.cell.2011.05.038. PMC 3124677. PMID 21703450.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Hara Y, Merten CA (2015). "Dynein-based accumulation of membranes regulates nuclear expansion in Xenopus laevis egg extracts". Dev Cell. 33 (5): 562–575. doi:10.1016/j.devcel.2015.04.016. PMID 26004509.
- ^ Pomerening JR, Kim SY, Ferrell JE Jr (2005). "Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations". Cell. 122 (4): 565–578. doi:10.1016/j.cell.2005.06.016. PMID 16122424. S2CID 11835940.
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