Biology:Xenopus egg extract

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Short description: Cell-free system

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

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

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

Figure 1. An egg extract is prepared by crushing X. laevis eggs by centrifugation

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.

Interphase (S phase) extract

Different types of egg extracts

Cycling extract

Figure 2. An interphase nucleus (left) and a cluster of mitotic chromosomes (right) produced in a cycling extract. Bar, 10 µm.

High-speed supernatant (HSS)

Nucleoplasmic extract (NPE)

Discoveries made using egg extracts

  • Purification of M-phase promoting factor (MPF)[6]
  • Elucidation of the role of synthesis and degradation of cyclin B in cell cycle progression[7][8]
  • Discovery that degradation of a protein(s) other than cyclin B is necessary for initiating chromosome segregation[9]
  • Discovery of a mechanism of spindle assembly that depends on chromatin, but not centrosomes[10]
  • Proposal of a DNA replication licensing system[11] and identification of its responsible factor[12]
  • Identification of importin α/β responsible for nuclear transport[13]
  • Discovery of the condensin complex essential for mitotic chromosome assembly[14][15]
  • Identification of the cohesin complex essential for sister chromatid cohesion[16]

More recently, the egg extracts have been used to study reprogramming of differentiated nuclei,[17] physical properties of spindles[18] and nuclei,[19] and theoretical understanding of cell cycle control.[20]

See also

References

  1. ↑ Lohka MJ, Masui Y (1983). "Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components". Science 220 (4598): 719–721. doi:10.1126/science.6601299. PMID 6601299. Bibcode: 1983Sci...220..719L. 
  2. ↑ 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. PMID 3926780. 
  3. ↑ 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. 
  4. ↑ 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. PMID 17339377. 
  5. ↑ 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. 
  6. ↑ 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. doi:10.1073/pnas.85.9.3009. PMID 3283736. Bibcode: 1988PNAS...85.3009L. 
  7. ↑ Murray AW, Kirschner MW (1989). "Cyclin synthesis drives the early embryonic cell cycle". Nature 339 (6222): 275–280. doi:10.1038/339275a0. PMID 2566917. Bibcode: 1989Natur.339..275M. 
  8. ↑ 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. doi:10.1038/339280a0. PMID 2566918. Bibcode: 1989Natur.339..280M. 
  9. ↑ 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. 
  10. ↑ 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. doi:10.1038/382420a0. PMID 8684481. Bibcode: 1996Natur.382..420H. 
  11. ↑ Blow JJ, Laskey RA (1988). "A role for the nuclear envelope in controlling DNA replication within the cell cycle". Nature 332 (6164): 546–548. doi:10.1038/332546a0. PMID 3357511. Bibcode: 1988Natur.332..546B. 
  12. ↑ 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. 
  13. ↑ 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. 
  14. ↑ 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. 
  15. ↑ 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. 
  16. ↑ 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. PMID 9649503. 
  17. ↑ 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. PMID 21908712. 
  18. ↑ 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. PMID 21703450. 
  19. ↑ 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. 
  20. ↑ 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. 



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