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High-quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics.

ABSTRACT: Cell-free extracts from unfertilized Xenopus laevis eggs offer the opportunity for a variety of biochemical and biophysical assays for analyzing essential cell cycle events such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, factors that hamper their experimental utility. Here we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels similar to those of freshly prepared extracts. Extract degradation associated with the freeze-thaw process can be substantially reduced by using centrifugal filter-based dehydration and slow sample cooling. Large amounts of frozen extract stocks from single-batch preparations allowed us to collect extensive data in micromanipulation experiments, which are often low-throughput, and thus enabled the clarification of correlations between metaphase spindle size and stiffness. Our method provides an assay platform with minimized biological variability and improves the accessibility of egg extracts for research.

SUBMITTER: Takagi J

PROVIDER: S-EPMC5531733 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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High-quality frozen extracts of <i>Xenopus laevis</i> eggs reveal size-dependent control of metaphase spindle micromechanics.

Takagi Jun J Shimamoto Yuta Y

Molecular biology of the cell 20170607 16

Cell-free extracts from unfertilized <i>Xenopus laevis</i> eggs offer the opportunity for a variety of biochemical and biophysical assays for analyzing essential cell cycle events such as metaphase spindle assembly. However, the extracts often exhibit substantial variation in quality and have low storage stability, factors that hamper their experimental utility. Here we report a simple two-step method for preparing frozen egg extracts that retain spindle assembly activity levels similar to those ...[more]

PMID: 28592634

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Project description:Predators can play an important role in regulating prey abundance and diversity, determining food web structure and function, and contributing to important ecosystem services, including the regulation of agricultural pests and disease vectors. Thus, the ability to predict predator impact on prey is an important goal in ecology. Often, predators of the same species are assumed to be functionally equivalent, despite considerable individual variation in predator traits known to be important for shaping predator-prey interactions, like body size. This assumption may greatly oversimplify our understanding of within-species functional diversity and undermine our ability to predict predator effects on prey. Here, we examine the degree to which predator-prey interactions are functionally homogenous across a natural range of predator body sizes. Specifically, we quantify the size-dependence of the functional response of African clawed frogs (Xenopus laevis) preying on mosquito larvae (Culex pipiens). Three size classes of predators, small (15-30 mm snout-vent length), medium (50-60 mm) and large (105-120 mm), were presented with five densities of prey to determine functional response type and to estimate search efficiency and handling time parameters generated from the models. The results of mesocosm experiments showed that type of functional response of X. laevis changed with size: small predators exhibited a Type II response, while medium and large predators exhibited Type III responses. Functional response data showed an inversely proportional relationship between predator attack rate and predator size. Small and medium predators had highest and lowest handling time, respectively. The change in functional response with the size of predator suggests that predators with overlapping cohorts may have a dynamic impact on prey populations. Therefore, predicting the functional response of a single size-matched predator in an experiment may misrepresent the predator's potential impact on a prey population.

Dataset Information

High-quality frozen extracts of Xenopus laevis eggs reveal size-dependent control of metaphase spindle micromechanics.

Publications

High-quality frozen extracts of <i>Xenopus laevis</i> eggs reveal size-dependent control of metaphase spindle micromechanics.

Similar Datasets

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