Abstract
The characteristic bipolar shape of the mitotic spindle is produced by the focusing of the minus ends of microtubules at the spindle poles. The focus is maintained by the centrosome, a microtubule-nucleating organelle, as well as by proteins that are capable of focusing kinetochore fibers (K fibers) even in the absence of a centrosome. Here, we have performed a small-scale RNA interference (RNAi) screen of known or suspected pole-related proteins in Drosophila S2 cells. An unexpected outcome of this screen was the finding that one of the four Drosophila Mob proteins (a family of kinase regulators) plays a role in spindle pole organization. Time-lapse microscopy of mitotic cells depleted of Drosophila Mob4 by RNAi revealed that the K fibers splay apart and do not maintain their focus either in the presence or absence of functional centrosomes. The Mob4 RNAi phenotype most closely resembles that observed after depletion of the protein encoded by abnormal spindle (Asp), although Asp localization is not substantially affected by Mob4 RNAi. Expression of a Drosophila Mob4-GFP fusion protein revealed its localization to the nucleus in interphase and to spindle poles and kinetochores during mitosis. We propose that Mob4 in Drosophila controls a mitotic kinase that in turn regulates downstream target proteins involved in K fiber focusing at the poles.
