Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Size specification of macromolecular assemblies in the cytoplasm is poorly understood [1]. In principle, assemblies could scale with cell size or use intrinsic mechanisms. For the mitotic spindle, scaling with cell size is expected, because the function of this assembly is to physically move sister chromatids into the center of nascent daughter cells. Eggs of Xenopus laevis are among the largest cells known that cleave completely during cell division. Cell length in this organism changes by two orders of magnitude ( approximately 1200 microm to approximately 12 microm) while it develops from a fertilized egg into a tadpole [2]. We wondered whether, and how, mitotic spindle length and morphology adapt to function at these different length scales. Here, we show that spindle length increases with cell length in small cells, but in very large cells spindle length approaches an upper limit of approximately 60 microm. Further evidence for an upper limit to spindle length comes from an embryonic extract system that recapitulates mitotic spindle assembly in a test tube. We conclude that early mitotic spindle length in Xenopus laevis is uncoupled from cell length, reaching an upper bound determined by mechanisms that are intrinsic to the spindle.
Figure 1. Spindle Size Is Uncoupled from Cell Size during First MitosesX. laevis embryos at various stages of development were fixed and stained for tubulin (yellow) and DNA (red).(A) Embryo at stage 8: animal pole with smaller cells and smaller spindles on top, vegetal pole with larger cells and larger spindles on bottom.(B) Embryo at mitosis 7, animal part.(C) Second mitotic spindle. White lines define spindle and cell size used throughout this paper.(D) Egg arrested at metaphase of meiosis II with arrow pointing at spindle. The scale bar for the upper row represents 500 μm. The scale bar for the lower row represents 20 μm.(E) Plot of spindle size versus cell size at different stages of development. Spindle size increases with cell size but asymptotically reaches an upper limit of ∼60 μm. Plot on the right is a zoom-in of smaller cells and spindles.
Figure 2. Embryo Extract Is Able to Assemble Mitotic SpindlesDNA is shown in red and tubulin in yellow.(A) Extract prepared from meiosis II-arrested eggs assembles spindles that show similar morphology to meiotic in vivo spindles.(B) Spindles in extract prepared from embryos were arrested in mitosis with addition of the APC inhibitor EmiI. The spindles formed show similar morphology to mitotic in vivo spindles. The scale bar represents 20 μm.
Figure 3. Halving the DNA Content Reduces Spindle Length by ∼10%(A) Percentage of embryos (synchronously fertilized) in anaphase (blue bars) was fitted to a cumulative Gaussian distribution (red line), and the time for metaphase-anaphase transition was calculated at 132 ± 3 min (SD) pf. Spindle length before peak of anaphase onset (full squares) was fitted linearly (green line), revealing spindle growth of 1.0 μm/min. Delayed spindles (shown as orange squares) were ignored for growth measurement because this would have systematically underestimated growth rate.(B) Albino eggs were fertilized with UV-treated sperm from a pigmented male, resulting in tadpoles with no pigments but haploid phenotype. Controls developed with pigments and diploid phenotype.(C) Sperm-derived DNA (arrow) is separate from spindles at two-cell stage of haploid embryo. The scale bar represents 500 μm.(D) Mean spindle length for haploids is 55.2 μm and therefore ∼10% shorter than diploids with 62.1 μm. Standard errors are 0.9 μm and 1.1 μm, respectively, with a statistically significant p value of 0.005%.
Figure 4. Relatively Small Spindle Is Compensated by Enormous Anaphase B-like MovementEmbryos of a synchronously fertilized population were fixed between first and second cytokineses and stained for tubulin (yellow) and DNA (red).(A) At anaphase the astral microtubules start to elongate.(B) Up to a DNA-to-DNA distance of ∼180 μm, DNA is still condensed and surrounded by high staining of microtubules. Astral microtubules form a hollow structure.(C) Nuclear envelope has reformed, and finally the nuclei have been separated by ∼400 μm, astral microtubules reach the cell cortex, and cytokinesis starts.(A–C) The scale bar for the upper row represents 500 μm; bars in lower row represent 20 μm.(D) Plot of DNA-to-DNA distance versus time. Linear fit estimates speed of DNA separation at ∼15 μm/min.(E) Cytokinesis, but not separation of DNA, is inhibited by addition of 33 μg/ml of actin-depolymerizing Cytochalasin B.
Andersen,
Mitotic chromatin regulates phosphorylation of Stathmin/Op18.
1997, Pubmed,
Xenbase
Andersen,
Mitotic chromatin regulates phosphorylation of Stathmin/Op18.
1997,
Pubmed
,
Xenbase
Becker,
Visualization of the cytoskeleton in Xenopus oocytes and eggs by confocal immunofluorescence microscopy.
2006,
Pubmed
,
Xenbase
Brown,
Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle.
2007,
Pubmed
,
Xenbase
Burbank,
Slide-and-cluster models for spindle assembly.
2007,
Pubmed
,
Xenbase
Desai,
The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.
1999,
Pubmed
,
Xenbase
Gard,
Confocal immunofluorescence microscopy of microtubules, microtubule-associated proteins, and microtubule-organizing centers during amphibian oogenesis and early development.
1995,
Pubmed
,
Xenbase
Gard,
F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation.
1995,
Pubmed
,
Xenbase
HAMILTON,
An experimental analysis of the development of the haploid syndrome in embryos of Xenopus laevis.
1963,
Pubmed
,
Xenbase
Heald,
Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.
1996,
Pubmed
,
Xenbase
Hirsch,
Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.
2002,
Pubmed
,
Xenbase
Lohka,
Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.
1985,
Pubmed
,
Xenbase
Marshall,
Cellular length control systems.
2004,
Pubmed
Mayr,
The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression.
2007,
Pubmed
Mitchison,
Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles.
2005,
Pubmed
,
Xenbase
Montorzi,
Xenopus laevis embryo development: arrest of epidermal cell differentiation by the chelating agent 1,10-phenanthroline.
2000,
Pubmed
,
Xenbase
Moses,
Cytostatic factor (CSF) in the eggs of Xenopus laevis.
1989,
Pubmed
,
Xenbase
Murray,
Cyclin synthesis drives the early embryonic cell cycle.
1989,
Pubmed
,
Xenbase
Nicklas,
The total length of spindle microtubules depends on the number of chromosomes present.
1985,
Pubmed
Noramly,
A gynogenetic screen to isolate naturally occurring recessive mutations in Xenopus tropicalis.
2005,
Pubmed
,
Xenbase
Reimann,
Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex.
2001,
Pubmed
,
Xenbase
Sagata,
The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs.
1989,
Pubmed
,
Xenbase
Sawai,
Cleavage plane determination in amphibian eggs.
1990,
Pubmed
,
Xenbase
Sawin,
Mitotic spindle assembly by two different pathways in vitro.
1991,
Pubmed
,
Xenbase
Schmidt,
Cytostatic factor: an activity that puts the cell cycle on hold.
2006,
Pubmed
,
Xenbase
Strauss,
A default mechanism of spindle orientation based on cell shape is sufficient to generate cell fate diversity in polarised Xenopus blastomeres.
2006,
Pubmed
,
Xenbase
Théry,
Experimental and theoretical study of mitotic spindle orientation.
2007,
Pubmed
Verlhac,
Asymmetric division in mouse oocytes: with or without Mos.
2000,
Pubmed
