Abstract
Author SummaryThe mitotic spindle is a bipolar structure that is responsible for separating the two sets of duplicated chromosomes in a dividing cell, thereby delivering one set to each of the two daughter cells. It is built from dynamic filaments called microtubules, as well as hundreds of other components that contribute to the organization and dynamics of the microtubules and to chromosome movement. To understand which proteins are essential for spindle formation and function, we would like to be able to build it from purified components. As a step towards this goal, we coupled individual proteins to inert glass beads (as a substitute for chromosomes), to determine what combination of proteins can induce spindle assembly in a complex cytoplasm derived from frog eggs. We found that a single enzyme called RCC1 is sufficient to activate a pathway that stabilizes and organizes microtubules into a bipolar structure around the bead, but that this bead then oscillated back and forth between the poles of the spindle. By coupling a microtubule-based motor protein together with RCC1 on the bead, we were able to balance the bead in the center of the spindle. Thus, two proteins immobilized on a bead can substitute for a chromosome and induce stable spindle formation.
Highlights
The spindle is a highly dynamic structure composed of microtubule polymers and hundreds of other factors including motor proteins and microtubule-associated proteins (MAPs) [1]
Addition of a hydrolysisdeficient mutant of Ran bound to GTP (RanQ69L-GTP) stabilizes microtubules that are organized by motor proteins into asters and small spindle-like structures in metaphase-arrested cytoplasmic extracts prepared from Xenopus laevis eggs [7,8,9,10]
RanQ69L-GTP disrupts the Regulator of Chromosome Condensation 1 (RCC1)-generated RanGTP gradient and spindle assembly [11], while flattening the gradient eliminates spindle assembly around chromatin beads [12]. These experiments demonstrate that a RanGTP gradient is required for chromatindependent spindle assembly in Xenopus egg extracts, but is it sufficient? We set out to test whether immobilized RCC1 in the absence of other chromatin factors can reconstitute a mitotic spindle
Summary
The spindle is a highly dynamic structure composed of microtubule polymers and hundreds of other factors including motor proteins and microtubule-associated proteins (MAPs) [1]. One candidate enzyme associated with chromatin that could drive spindle assembly is RCC1, the guanine nucleotide exchange factor (GEF) for the small GTPase Ran. RCC1 generates a steep gradient of RanGTP near chromosomes, activating a subset of mitotic motors and MAPs that are cargoes of the importin b family of nuclear transport receptors [6]. RanQ69L-GTP disrupts the RCC1-generated RanGTP gradient and spindle assembly [11], while flattening the gradient eliminates spindle assembly around chromatin beads [12]. These experiments demonstrate that a RanGTP gradient is required for chromatindependent spindle assembly in Xenopus egg extracts, but is it sufficient? These experiments demonstrate that a RanGTP gradient is required for chromatindependent spindle assembly in Xenopus egg extracts, but is it sufficient? We set out to test whether immobilized RCC1 in the absence of other chromatin factors can reconstitute a mitotic spindle
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