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, 172 (3), 363-72

KIF14 and Citron Kinase Act Together to Promote Efficient Cytokinesis

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KIF14 and Citron Kinase Act Together to Promote Efficient Cytokinesis

Ulrike Gruneberg et al. J Cell Biol.

Abstract

Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis.

Figures

Figure 1.
Figure 1.
The central spindle MAP PRC1 interacts with multiple kinesins. (A) Proteins immune precipitated from 10 mg of anaphase HeLa cell extract with affinity-purified rabbit antibodies to GST or PRC1 were separated on minigels and identified by mass spectrometry. The immunoglobulin G heavy (IgH) and light chains (IgL) are marked, and the asterisk denotes a nonspecific contaminant found with both control and KIF14 antibodies. (B) Lysates from HeLa cells treated with control or PRC1 siRNA duplexes for 36 h were Western blotted for PRC1 and α-tubulin as a loading control. (C) GST and KIF14 immune precipitates were Western blotted for KIF14 and PRC1. (D) HeLa cells treated with control or PRC1 siRNA duplexes for 30 h were fixed and stained with antibodies to PRC1 or KIF14 (red) and α-tubulin (green). DNA was stained with DAPI (blue). Bars, 10 μm.
Figure 2.
Figure 2.
Depletion of KIF14 results in a failure of cytokinesis. (A) Lysates from HeLa cells treated with control or KIF14 siRNA duplexes for 48 h were Western blotted for KIF14 and α-tubulin as a loading control. (B and C) HeLa cells treated with control or KIF14 siRNA duplexes for 48 h were fixed and stained with antibodies to KIF14 (red) and α-tubulin (green). (C) The number of binucleated cells was counted and is expressed as a percentage in control and KIF14-depleted cells. A typical field of KIF14-depleted cells from such an experiment is shown. DNA was stained with DAPI (blue). Bars, 10 μm.
Figure 3.
Figure 3.
KIF14 localizes to the central spindle and midbody. HeLa cells expressing EGFP-KIF14 (green) were fixed and stained for KIF14 (green) and PRC1 (red) 24 h after transfection. DNA was stained with DAPI (blue). Bar, 10 μm.
Figure 4.
Figure 4.
Central spindle and midbody targeting of KIF14 involves the amino-terminal extension. (A and B) A schematic of human KIF14 indicating the unique extension amino terminal of the kinesin motor domain and the forkhead-associated (FHA) domain that defines the kinesin-3 family of motor proteins. HeLa cells were transfected with EGFP-tagged constructs encoding full-length KIF14 and the subdomains indicated in the table. Cells were fixed and stained for α-tubulin. The localizations of these proteins in interphase and mitotic HeLa cells is summarized in the table. (B) The localization of the KIF14 amino-terminal extension amino acids 1–356 (green) and α-tubulin (red) are shown in interphase, telophase, and in cells undergoing cytokinesis. DNA was stained with DAPI (blue). Bar, 10 μm. (C) Immune precipitations (IPs) were performed using GFP antibodies from HEK293T cells transfected for 40 h with GFP or GFP-tagged KIF14 constructs as indicated in the figure. Samples of the lysate and IP were analyzed by Western blotting for GFP and PRC1. The asterisk indicates a premature termination or possible breakdown product seen with all amino-terminal fragments of KIF14.
Figure 5.
Figure 5.
KIF14 is required for the localization of citron kinase but not other components of the central spindle or cleavage furrow. HeLa cells transfected with control or KIF14 siRNA duplexes for 48 h were fixed and stained with antibodies to the proteins (red) indicated in the figure. Actin was detected using 100 ng/ml rhodamine-phalloidin (red). DNA was stained with DAPI (blue). Bar, 10 μm. Arrows mark the positions of the cleavage furrow in control and KIF14-depleted cells stained for citron kinase.
Figure 6.
Figure 6.
KIF14 interacts with PRC1 and citron kinase. Immune precipitations from anaphase HeLa cell extracts were performed using the antibodies indicated across the top of the figure and described in Materials and methods. The input lane represents 10% of material used for each IP. The immune precipitates were then separated by SDS-PAGE and analyzed by Western blotting with the antibodies indicated down the left side of the figure.
Figure 7.
Figure 7.
Interaction of citron kinase with KIF14 is dependent on its kinase activity. (A and C) IPs were performed using FLAG antibodies from HEK293T cells cotransfected for 40 h with wild-type (WT; A) or kinase-dead (KD) FLAG-tagged citron kinase and GFP-tagged KIF14 or INCENP constructs as indicated in the figure. Samples of the lysates and FLAG IP were analyzed by Western blotting for GFP and the FLAG epitope. (B) IPs were performed using FLAG antibodies from HEK293T cells transfected for 40 h with wild-type or kinase-dead FLAG-tagged citron kinase. Samples of the lysates and FLAG IP were analyzed by Western blotting for KIF14 and the FLAG epitope. (C) The running positions of the various KIF14 constructs are indicated to the right of the top panel. KIF14 constructs that efficiently coprecipitated with citron kinase are indicated to the right of the middle panel.
Figure 8.
Figure 8.
Citron kinase is required for cytokinesis and for the localization of KIF14 to the midbody. (A) Cell lysates from HeLa cells treated with control or citron kinase (CK) siRNA duplexes for 50 h were Western blotted for citron kinase, KIF14, and α-tubulin as a loading control. (B and C) HeLa cells treated with control or citron kinase siRNA duplexes for 50 h were fixed and stained with antibodies to citron kinase (green) and KIF14 (red). (B) A typical field of KIF14-depleted cells from such an experiment is shown. The number of binucleated cells was counted and is expressed as a percentage in control and citron kinase–depleted cells. (C) Localization of KIF14 in control and citron kinase–depleted cells. DAPI, blue. (D) HeLa cells treated with control, citron kinase, or KIF14 siRNA duplexes for 50 h were fixed and stained with antibodies to MKlp1 (red) and citron kinase or tubulin (green). Arrows on the merged images indicate midbody remnants. Bars, 10 μm.
Figure 9.
Figure 9.
PRC1 forms a central spindle matrix directing kinesin function during cytokinesis. PRC1 is a master regulator of the central spindle interacting with multiple motor proteins that are involved in cytokinesis. Centralspindlin, shown as MKlp1 for simplicity, controls Rho activation and inactivation, whereas KIF14 is needed for citron kinase localization to the central spindle, midbody, and, thus, its site of activity. One function of Rho is to control the activation state of citron kinase. MKlp2 is needed for both the targeting of Plk1 and the chromosomal passenger complex and, thus, for spatial control of both Plk1 and Aurora B. These kinases phosphorylate proteins required for cytokinesis, including components of the centralspindlin complex. The molecular function of KIF4 is less clear, but it too appears to be required for cytokinesis. In the absence of MKlp1 and MKlp2, early events in central spindle and cleavage furrow formation are abnormal. This leads to defects in furrow positioning and contractility. KIF14 and citron kinase are not required for these early events or late events such as midbody formation but are needed for efficient cytokinesis.

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