Assembly of Spindles and Asters in Xenopus Egg Extracts.

Abstract

Here, we provide methods for assembly of mitotic spindles and interphase asters in Xenopus laevis egg extract, and compare them to spindles and asters in the egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) extract and inducing entry into interphase extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes. CSF spindles are made by addition of sperm nuclei to CSF extract. They are less physiological and lack functional kinetochores but suffice for some applications. Large interphase asters are prepared by addition of artificial centrosomes or sperm nuclei to actin-intact egg extract. These asters grow rapidly to hundreds of microns in radius by branching microtubule nucleation at the periphery, so the aster as a whole is a network of short, dynamic microtubules. They resemble the sperm aster after fertilization, and the asters that grow out of the poles of the mitotic spindle at anaphase. When interphase asters grow into each other they interact and assemble aster interaction zones at their shared boundary. These zones consist of a line (in extract) or disc (in zygotes) of antiparallel microtubule bundles coated with cytokinesis midzone proteins. Interaction zones block interpenetration of microtubules from the two asters, and signal to the cortex to induce cleavage furrows. Their reconstitution in extract allows dissection of the biophysics of spatially regulated cytokinesis signaling.