Methods to analyze novel liaisons between endosomes and centrosomes

Abstract

For some time, it has been known that recycling endosomes (REs) are organized in a nebulous "pericentrosomal" region in interphase cells. However, the collective use of previously developed methods, including centrosome isolation, live cell imaging, and electron microscopy, suggested that there is much more going on between the centrosome and the RE than previously imagined. By exploiting these approaches, we uncovered novel roles of the centrosome in RE function and, conversely, novel roles for REs in centrosome function. We first found that REs dynamically localized to the centrosome throughout the cell cycle. More specifically, we found that REs interacted with appendages of the older centriole in interphase cells to control endosome recycling, and this interaction was governed by RE-machinery including the small GTPase Rab11. We next determined that REs carry centrosome proteins to spindle poles as part of the "centrosome maturation" process. Here we discuss the methods used and materials needed to complete these types of studies.

Keywords: Appendages; Cenexin; Centriolin; Centrosome; Centrosome isolation; Evi5; Fip3; Mitosis; Mother centriole; Rab11; Rab8; Recycling endosome; Spindle pole.