Encapsulation of Xenopus egg and embryo extract spindle assembly reactions in synthetic cell-like compartments with tunable size

Abstract

Methods are described for preparing Xenopus laevis egg and embryo cytoplasm and encapsulating extract spindle assembly reactions in cell-like compartments to investigate the effects of cell size on intracellular assembly. Cytoplasm prepared from the eggs or embryos of individual frogs is screened for the ability to form interphase nuclei and metaphase spindles, and subsequently packaged, along with DNA, into droplets of varying size using microfluidics. The dimensions of these cell-like droplets are specified to match the range of cell diameters present in early embryo development. The scaling relationship between droplets and spindles is quantified using live fluorescence imaging on a spinning-disk confocal microscope. By comparing the encapsulated assembly of spindles formed from cytoplasmic extracts prepared from embryos at distinct stages of Xenopus early development, the influence of cell composition and cell size on spindle scaling can be evaluated. Because the extract system is biochemically tractable, the function of individual proteins in spindle scaling can be evaluated by supplementing or depleting factors in the cytoplasm.