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The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were approximately 30% shorter than X. laevis spindles, and mixing experiments revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. Measurements of microtubule dynamics revealed that microtubules polymerized slower in X. tropicalis extracts compared to X. laevis, but that this difference is unlikely to account for differences in spindle size. Thus, in addition to expanding the range of developmental and cell biological experiments, the use of X. tropicalis provides novel insight into the complex mechanisms that govern spindle morphogenesis.
Figure 1. X. tropicalis egg extracts recapitulate major cell cycle events in vitro. (A) CSF-arrested egg extracts prepared from eggs of X. tropicalis and supplemented with X. laevis sperm nuclei and X-rhodamineâlabeled tubulin formed âhalf spindlesâ in metaphase-arrested extracts, interphase nuclei after release from CSF arrest, and bipolar spindles when induced to reenter metaphase. Microtubules are red and DNA is blue. (B) X. tropicalis extract reactions were stained with fluorescently labeled antibody (α-H1 and α-NuMA), or by immunofluorescence (α-Xkid) using antibodies raised to the X. laevis proteins. Staining patterns recapitulated those in X. laevis reactions. In overlays, the stained protein is green. (C) CSF extracts pooled from eggs of multiple X. laevis or X. tropicalis frogs were blotted for Rae1, RCC1, and histone H1. In X. laevis extracts, all proteins gave multiple bands, presumably because of multiple genes for each protein, whereas X. tropicalis contained single isoforms. Bars, 10 μm.
Figure 2. Identification of X. tropicalis and X. laevis Xnf7 by mass spectrometry. (A) Xnf7 was immunoprecipitated from X. laevis or X. tropicalis extracts and subjected to SDS-PAGE. Excised bands were processed for MALDI-TOF mass spectrometry. (B) Xnf7-derived peptide masses from each band were compared against databases from both Xenopus species. The number of peptides that showed a perfect match against each database is shown. (C) Schematic representation of the Xnf7 domain structure showing the number of identical/conserved amino acids in each domain. Domains are in regular font and linker regions are in italics.
Figure 3. Comparison of spindle length between X. tropicalis and X. laevis. (A) Spindles assembled around X. laevis sperm nuclei in either X. laevis or X. tropicalis egg extracts were visualized using Hoechst dye (blue, DNA) and the incorporation of X-rhodamine tubulin (red microtubules, X. tropicalis), or Alexa Fluor 488 tubulin (green microtubules, X. laevis). Bar, 10 μm. (B) Mixed reactions with the indicated proportion of X. tropicalis extract were combined with X. laevis or X. tropicalis sperm nuclei. Spindle length was measured from pole to pole. A linear relationship was observed between the proportion of X. laevis extract present and spindle length. Error bars are the SD.
Figure 4. Spindle length determination is highly dynamic in Xenopus extracts. (A) Spindles assembled in X. tropicalis extracts containing X-rhodamine tubulin were diluted with three volumes of X. laevis or X. tropicalis extract supplemented with Alexa Fluor 488 tubulin, and fixed for examination at various time points. Quantification of spindle length over time after mixing revealed steady-state spindle lengths were reached within â¼5 min, and they corresponded to those in the premixed control of 75% X. laevis 25% X. tropicalis extract. (B) The same experiment described in A, except that the spindles were assembled in X. laevis extract containing Alexa Fluor 488 tubulin, and diluted with X. tropicalis or X. laevis extract containing X-rhodamine tubulin. Quantification of spindle length over time after mixing is shown. Error bars are the SDs. Bars, 10 μm.
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