The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization

Abstract

A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly.

SUPPLEMENTAL MATERIAL

We thank Praise Opara for technical assistance with zebrafish maintenance and cell culture, K. Rose Finley and James Ziai for technical support with the haploid screening, Matthew Keefe for embryo microinjections, Bruce Barut for assistance with the positional cloning project, Susannah Rankin and Marc Kirschner for reagents and advice on the mammalian cell experiments, Marnie Halpern and Joseph Yost for plasmids, and Craig Ceol for reagents and advice on the antibody production. We also thank Jim Amatruda, Caroline Burns, Alan Davidson, Michael Dovey, David Langenau, Ryan Murphey, Jennifer Shepard, and Howard Stern for helpful conversations throughout the course of this work. We are grateful to Craig Ceol, Jenna Galloway, Marc Kirschner, Trista North, and David Pellman for critical reading of the manuscript.

This work was supported by National Institutes of Health grant 5R01 CA103846-02 (to L.I.Z) and an Albert J. Ryan Fellowship (to K.L.P). L.I.Z. is an Investigator of the Howard Hughes Medical Institute.