Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay

Abstract

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells.

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