Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

Abstract

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

Molecular conflicts disrupting centromere assembly contribute to Xenopus hybrid inviability

“…We previously observed chromosome mis-segregation and loss of centromere and kinetochore proteins from a subset of chromosomes in hybrids generated by fertilizing X. tropicalis eggs with X. laevis sperm. Whole genome sequencing just prior to embryo death revealed consistent deletion of large genomic regions from two paternal chromosomes, 3L and 4L (Gibeaux et al, 2018). We hypothesized that chromosome-specific aneuploidy resulted from divergent centromeric sequences on the affected chromosomes, rendering them incompatible with the maternal X. tropicalis centromeric histone CENP-A and its loading machinery.…”

“…To expand our analysis, we characterized a second inviable hybrid resulting from fertilization of X. tropicalis eggs with sperm from X. borealis, a frog species possessing an allotetraploid genome closely related to X. laevis (Session et al, 2016). These hybrids display specific and consistent genome loss from a different subset of paternal chromosomes including 1S, 5S, 4L, and 8L (Gibeaux et al, 2018). To determine the extent to which centromere sequences differed across the three Xenopus species, CENP-A ChIP-seq was similarly applied to X. tropicalis and X. borealis.…”

Affinity Purification of Label-free Tubulins from Xenopus Egg Extracts

“…( Brown et al., 2007 ) and X. borealis ( X.b. ) ( Kitaoka et al., 2018 ). Note: Yields of extract and therefore tubulin will depend on egg quantity and size ( Table 1 ).…”

“…are 1.3±0.05 mm in diameter, similar to X.b. eggs with a diameter of 1.2±0.02 mm ( Kitaoka et al., 2018 ). In either case, a batch of eggs from one frog will yield ∼1 mL of extract.…”

Xenopus Hybrids Provide Insight Into Cell and Organism Size Control

“…However, the molecular mechanisms that control size at the organism, cell, and subcellular levels are poorly understood. The frog Xenopus has emerged as a powerful system to explore nuclear and spindle size differences that occur between related species with different-sized eggs (Levy and Heald, 2010; Loughlin et al, 2011; Kitaoka et al, 2018), as well as subcellular scaling during early development, when cleavage divisions cause a rapid reduction in cell size (Good et al, 2013; Wilbur and Heald, 2013). We therefore set out to investigate whether Xenopus frogs could also be used to study size control at the level of the cell and the whole organism.…”