Kinesin 5B (KIF5B) is required for progression through female meiosis and proper chromosomal segregation in mitotic cells

Abstract

The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

Figure 1. Downregulation of KIF5B blocks Metaphase I in female meiosis.

A) Western blot analysis of oocytes treated with siRNA against Kif5b or control (luciferase) B) Live images of oocytes at different times after microinjection with control-RNAi and Kif5b-RNAi. C) Kinetics of GVBD in oocytes at different time points. D) Estimated percentage of polar body extrusion (PBE) (control (n = 44) and Kif5b-RNAi (n = 45).

Figure 2. Characterization of mouse KIF5B during cell division.

A) Schematic diagram of KIF5B domains based http://www.ensembl.org sequence database. Blue, red, cyan and green boxes indicate motor, nucleotide binding, coiled-coil and globular domains respectively. B) Dynamic localization of KIF5B during different stages of cell division. Note that red and green represent DNA and KIF5B respectively.

Figure 3. Downregulation of KIF5B leads to chromosome misalignment.

A) Western blot showing the efficiency of Kif5b-shRNA-mediated knockdown with whole- cell extracts prepared from HeLa and control cells transfected with control-shRNA. B) Cell division-dependent localization of microtubules (green) and DNA arrangement (red) in Kif5b-shRNA versus control. Note that the arrow indicates examples of tripolar arrangement in microtubules. C) A statistically significant difference was observed for the estimated percentage of cells with multipolar arrangements of microtubules in Kif5b-shRNA knockdown cells (n = 110) versus wild type (n = 98)(*** p<0.0001). The Graph Pad Prism program was used for data analysis employing Mann Whitney statistical methods.

Figure 4. KIF5B is required for chromosome segregation and cytokinesis.

A) Examples of control cells with normal anaphase chromosome separation (n = 50). B) Anaphase bridges shown with arrow in Kif5b downregulated cells (n = 40). Note that *** represents P<0.0001 statistically significant differences between control cells that are transfected with scrambled shRNA and Kif5b knockdown cells in chromosome arrangements at anaphase stage. C) Representative images of cells with normal and abnormal cytokinesis. D) The percentage of cells with cytokinesis failure is significantly increased (* P<0.01) in Kif5b-shRNA-mediated knockdown cells (n = 77) compared to control cells transfected with scrambled shRNA cells (n = 109). Note that the white arrow points to an example of aberrant cytokinesis, red represents DNA, and green is α- tubulin.

Figure 5. KIF5B suppresses centrosome amplification.

A–C) Control cells during different stages of cell division with one and two centrosomes. D–F) Representative images of cells with Kif5b-shRNA-mediated knockdown with more than two centrosomes. The DNA is stained with DAPI (blue), and centrosomes are stained with γ-tubulin (green). The arrow indicates the centrosome in each cell. G) The representative number of cells analyzed for centrosomes in control cells (n = 119) and Kif5b-shRNA knockdown cells (n = 175). Cells with greater than two centrosomes were significantly enriched by Kif5b-shRNA-mediated knockdown (*** P<0.0001). The Graph Pad Prism program was used for analysis of the data and employed the Mann Whitney test.