Small GTPase Rab5 participates in chromosome congression and regulates localization of the centromere-associated protein CENP-F to kinetochores

Abstract

Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.

A new role for Rab GTPases during early mitotic stages

“…In Drosophila, proteomic analysis of Rab5 interacting partners identified MUD (mushroom body defect), the Drosophila homolog of NuMA, further supporting the role of Rab5 in spindle maturation as seen for Rab11. 9,22,23 These observations, suggest unique liaisons between early endosomes (Rab5-labeled) and recycling endosomes (Rab11-labeled) during mitosis. For example, in interphase cells Rab5 endosomes can mature into Rab11 endosomes.…”

“…1) and plays a role in mitotic progression through the regulation of the spindle maturation protein, NuMA, 22 and the kinetochore protein, CENP-F(centrosome associated protein). 23 Strikingly similar to disruption of Rab11, 9 Rab5-depletion caused numerous mitotic-defects such as chromosome misalignment, delays in metaphase and anaphase progression, 22 and defects in cytokinesis that resulted in multinucleated cells. 22,23 Again similar to Rab11, 9 Rab5 localized to endosomes around spindle poles 22 ( Fig.…”

“…23 Strikingly similar to disruption of Rab11, 9 Rab5-depletion caused numerous mitotic-defects such as chromosome misalignment, delays in metaphase and anaphase progression, 22 and defects in cytokinesis that resulted in multinucleated cells. 22,23 Again similar to Rab11, 9 Rab5 localized to endosomes around spindle poles 22 ( Fig. 1), as demonstrated by the colocalization of GFP-Rab5 with FM4-64-stained membranes and the global endosomal marker, GFP::2xFYVE.…”

“…23 Mitotic phenotypes associated with Rab5-depletion (e.g., instability of kinetochore fibers, chromosome misalignment, etc.) were similar to CENP-F depletion, suggesting a tight relationship that requires further elucidation.…”

A novel function of Rab5 in mitosis

“…Recently, we and others 1,2 found that a key player in endocytosis, the small GTPase Rab5, controls the alignment of chromosomes on the metaphase plate both in mammalian cells and in the Drosophila model organism. Although the underlying mechanisms appear to be distinct, depletion of Rab5 affects progression through mitosis and the correct execution of chromosome segregation in the daughter cells in both systems, indicating that this function of Rab5 is conserved through evolution.…”