The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK

Abstract

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore-microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle bipolarity. These treatments failed to restore bipolarity to cells lacking the activity of the kinesin Eg5. Thus, two independent pathways contribute to spindle bipolarity, with the Eg5-dependent pathway using motor force to drive spindle bipolarity and the Kif2a-dependent pathway relying on microtubule polymer dynamics to generate force for spindle bipolarity.

Figure 1.

Characterization of Kif2a in cultured vertebrate cells. (A) Total cell protein from ∼100,000 human HeLa, frog A6, or hamster CHO cells was separated by SDS-PAGE and blotted with Kif2a polyclonal antibodies. Positions of myosin (200), β-galactosidase (116), phosphorylase B (97), albumin (66), and ovalbumin (45) are shown in kilodaltons. (B–F) Human CFPAC-1 cells were processed for indirect immunofluorescence microscopy using Kif2a- (red) and tubulin-specific antibodies (green), as well as the DNA-specific dye DAPI (blue). Cells are in interphase (B), prophase (C), prometaphase (D), metaphase (E), and anaphase (F). Bar, 10 μm.

Figure 2.

Kif2a is essential for spindle bipolarity. (A) Human U2OS cells were transfected with either nonspecific (control) or Kif2a-specific (siRNA) RNAs. Total cell protein was separated by size using SDS-PAGE and blotted with Kif2a-specific antibodies and tubulin antibodies. (B–G) Human U2OS cells were transfected with either nonspecific (control) or Kif2a-specific (siRNA) RNAs, fixed with either glutaraldehyde (B and C) or methanol (D–G), and stained for tubulin (green), DNA (blue), and/or Kif2a (red). Bars, 10 μm.

Figure 3.

Nocodazole treatment restores spindle bipolarity to cells lacking Kif2a. U20S cells stained for tubulin (green) and DNA (blue) after transfection with Kif2a-specific RNA (A and C) or treatment with 100 μM monastrol (B and D) in the presence (C and D) or absence (A and B) of 100 nM nocodazole. Bar, 10 μm.

Figure 4.

MCAK knockdown restores spindle bipolarity to cells lacking Kif2a. (A) U2OS cells were transfected with nonspecific (C), Kif2a-specific (-K), MCAK-specific (-M), or both Kif2a- and MCAK-specific (-KM) RNAs. Total cell protein was separated by size using SDS-PAGE and blotted with Kif2a-, MCAK-, and tubulin-specific antibodies. (B–E) Human U2OS cells were stained for tubulin (green) or DNA (blue) after transfection with Kif2a-specific RNA alone (B), transfection with both Kif2a- and MCAK-specific RNA (D), treatment with 100 μM monastrol alone (C), or transfection with MCAK-specific RNA and treatment with 100 μM monastrol (E). Bar, 10 μM.

Figure 5.

Nuf2 knockdown restores spindle bipolarity to cells lacking Kif2a. (A) U2OS cells were transfected with nonspecific (C), Kif2a-specific (-K), Nuf2-specific (-N), or both Kif2a- and Nuf2-specific (-KN) RNAs. Total cell protein was separated by size using SDS-PAGE and blotted with Kif2a-, Hec1-, and tubulin-specific antibodies. (B–E) Human U2OS cells were stained for tubulin (green) or DNA (blue) after transfection with control RNA (B), Kif2a-specific RNA (D and E), Nuf2-specific RNA (C, E, and G), or treatment with 100 μM monastrol (F and G). Bar, 10 μM. (H and I) Quantification of the percentage of mitotic U2OS cells with bipolar spindles under various conditions after Kif2a knockdown (H) or monastrol treatment (I). Error bars represent SD. (J) Kif2a depolymerizes microtubule minus ends associated with poleward microtubule flux. Flux generates tension that induces kinetochores to add tubulin subunits. When flux is perturbed in the absence of Kif2a, tension at kinetochores is lost and kinetochores depolymerize microtubules drawing centrosomes together into monopolar arrays.