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Lte1 Promotes Mitotic Exit by Controlling the Localization of the Spindle Position Checkpoint Kinase Kin4

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Lte1 Promotes Mitotic Exit by Controlling the Localization of the Spindle Position Checkpoint Kinase Kin4 Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4 Jill E. Falk1, Leon Y. Chan1,2, and Angelika Amon3 David H. Koch Institute for Integrative Cancer Research and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2010. Contributed by Angelika Amon, May 17, 2011 (sent for review April 27, 2011) For a daughter cell to receive a complete genomic complement, it is The work by Adames et al. (13) proposed a model in which essential that the mitotic spindle be positioned accurately within the interactions between microtubules and the bud neck inhibit the cell. In budding yeast, a signaling system known as the spindle MEN, but how this could lead to Kin4 activation, if indeed Kin4 is position checkpoint (SPOC) monitors spindle position and regulates activated by spindle misposition, is not known. We previously the activity of the mitotic exit network (MEN), a GTPase signaling proposed a model termed the zone model, which posits that the pathway that promotes exit from mitosis. The protein kinase Kin4 budding yeast cell is divided into a MEN inhibitory zone in the is a central component of the spindle position checkpoint. Kin4 mother cell and a MEN activating zone in the daughter cell and primarily localizes to the mother cell and associates with spindle that a sensor, the GTPase Tem1, moves between them. Tem1 as well as most other components of the MEN reside at pole bodies (SPBs) located in the mother cell to inhibit MEN spindle pole bodies (SPBs; yeast centrosomes). An activator of signaling. In contrast, the kinase does not associate with the SPB the MEN, Lte1, localizes to the bud; the MEN inhibitor, Kin4, is in the bud. Thus, only when a MEN bearing SPB leaves the mother – enriched in the mother cell and at the mother SPB (4, 7, 14, 15). cell and the spindle is accurately positioned along the mother bud The asymmetric localization of these two key Tem1 regulators axis can MEN signaling occur and cell division proceed. Here, we allows the cell to trigger the MEN only when at least one SPB has describe a mechanism ensuring that Kin4 only associates with escaped the zone of inhibition in the mother cell, where Kin4 mother cell-located SPBs. The bud-localized MEN regulator Lte1, resides, and enters the zone of activation in the bud where Lte1 whose molecular function has long been unclear, prevents Kin4 that resides (Fig. 1A). When the spindle is mispositioned, neither SPB escapes into the bud from associating with SPBs in the daughter cell. escapes the mother cell, and the MEN remains inhibited (Fig. 1B). Importantly, in the zone model, spindle position is coupled to olarized cell division or the division of the cell along a pre- MEN activation during every cell cycle and is not a regulatory determined axis is critical to building higher-order biological mechanism that is only elicited in response to spindle misposition. P fi structures. Eukaryotic cells that divide in this manner must po- Although the role of Kin4 in MEN regulation is well-de ned, sition the mitotic spindle along this defined axis to ensure that the role of Lte1 is not clear. The protein is essential for exit from each mitotic product receives a complete genomic copy. The mitosis at low temperatures and has homology to guanine nucle- spindle position checkpoint (SPOC) is a feedback mechanism otide exchange factors (GEFs). Despite this homology, Lte1 GEF that delays cell-cycle progression in response to defects in spindle activity has, however, not been detected in vitro (16, 17). Here, position, and it has been found to be operative during the division prompted by the observation that when Kin4 and Lte1 reside in the of budding yeast, fruit fly stem cells, and cultured mammal- same compartment, Lte1 activation of the MEN prevails over Kin4 – inhibition (4, 17–19), we investigate the possibility that Lte1 ian cells (1 3). This surveillance mechanism is best understood in fi budding yeast, where every cell division is polarized and requires inhibits Kin4. Indeed, we nd that Lte1 prevents Kin4 from as- proper positioning of the mitotic spindle along the mother–bud sociating with the SPB in the daughter cell during anaphase. Our axis to produce euploid daughter cells. The SPOC prevents exit results indicate that a key aspect of spindle position surveillance is from mitosis when the spindle is not aligned along the mother– to allow Kin4 to inhibit MEN signaling from SPBs that reside in bud axis (1). When the SPOC is defective, cells with misposi- the mother cell but not from SPBs that reside in the daughter cell. At least two control mechanisms ensure that this finding is the tioned spindles inappropriately exit from mitosis, giving rise to i ii abnormal mitotic products with either zero or two nuclei (4–7). case: ( ) Kin4 is restricted to the mother cell, and ( )bud- In cells with mispositioned spindles, the SPOC delays cell- restricted Lte1 inhibits any Kin4 that enters the bud from loading cycle progression by inhibiting the mitotic exit network (MEN). onto the SPBs in the bud. By these mechanisms, inappropriate This pathway triggers exit from mitosis by bringing about the inhibition of mitotic exit by Kin4 is prevented. inactivation of mitotic cyclin-dependent kinases (CDKs), which Results are responsible for promoting entry into and progression through mitosis (reviewed in ref. 8). MEN signaling commences with the LTE1 Prevents Kin4 from Associating with SPBs in the Bud. Genetic LTE1 KIN4 activation of the MEN GTPase Tem1. The GTPase then signals studies have placed upstream of or in parallel to in lte1Δ through a kinase cascade to activate the protein phosphatase, regulating the MEN. The mitotic exit defect of cells is Cdc14, the major antagonist of mitotic cyclin-CDK activity. The SPOC controls mitotic exit by regulating the activation of Tem1. When the spindle is mispositioned, the SPOC kinase, Author contributions: J.E.F., L.Y.C., and A.A. designed research; J.E.F. and L.Y.C. per- Kin4, activates the GTPase-activating protein complex (GAP) of formed research; J.E.F., L.Y.C., and A.A. analyzed data; and J.E.F., L.Y.C., and A.A. wrote Tem1, the Bub2-Bfa1 complex, through phosphorylation of Bfa1 the paper. on residues S150 and S180 (9). Kin4 itself is regulated at multiple The authors declare no conflict of interest. levels. For example, phosphorylation by the protein kinase Elm1 1J.E.F. and L.Y.C. contributed equally to this work. is critical for kinase activity, whereas the protein phosphatase 2A 2Present address: Department of Molecular and Cell Biology and QB3, University of bound to its targeting subunit Rts1 regulates Kin4 localization California, Berkeley, CA 94720-3200. (10–12). 3To whom correspondence should be addressed. E-mail: [email protected]. How does the SPOC sense spindle position and translate this This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. spatial information into a chemical signal to regulate the MEN? 1073/pnas.1107784108/-/DCSupplemental. 12584–12590 | PNAS | August 2, 2011 | vol. 108 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1107784108 Downloaded by guest on October 1, 2021 AB for Lte1 in promoting mitotic exit came from the observation that when Lte1 and Kin4 are in the same compartment—either by INAUGURAL ARTICLE targeting Lte1 to the mother cell or by targeting Kin4 to the bud— Lte1 activation of the MEN prevails over Kin4 inhibition of this signaling pathway. These findings raise the possibility that Lte1 is an inhibitor of Kin4. Because Kin4 inhibits MEN signaling at SPBs (9), we first tested this hypothesis by examining the effects of deleting LTE1 on Kin4 localization. C Kin4 localization is dynamic. In G1 cells, the protein localizes to the cell cortex. When cells enter the cell cycle, the protein transiently localizes to the emerging bud. Shortly thereafter, Kin4 predominantly localizes to the mother cell cortex. During late anaphase, the protein also binds the SPB in the mother cell (mSPB). The fact that Kin4 associates with the mSPB only during late and not during early anaphase is reflected in the quantifica- tion of Kin4 localization in anaphase cells. Only 40–50% of anaphase cells harbor Kin4 at mSPBs (15) (Figs. 1 C and D,2B, 3B, and 4B). Shortly before cytokinesis, mother cortex enrich- ment of Kin4 becomes less pronounced, and Kin4 is also seen at D the bud neck and adjacent bud cortex (14, 15, 19). To determine whether LTE1 affects Kin4 localization, we analyzed the localization of the protein kinase in cells lacking LTE1. Deletion of LTE1 did not seem to affect the association of Kin4 with the mother cell cortex or the mSPB. However, whereas Kin4 was only detected on the daughter SPB (dSPB) in 2.0 ± 1.1% (SEM) of WT cells during anaphase, the protein was detected on the dSPB in 47.3 ± 4.1% (SEM) of lte1Δ cells (Fig. 1 E C and D). Kin4 loading onto the dSPB in lte1Δ mutants was not caused by the cell-cycle delay displayed by cells lacking LTE1 CELL BIOLOGY (19, 20). Cells that lack both LTE1 and BUB2 exit mitosis with WT kinetics (4, 13, 15, 20, 21), and in these cells, Kin4 was still observed to load onto dSPBs, albeit with reduced efficiency (Fig. 2 A and B). Taken together, these data indicate that LTE1 prevents Kin4 from loading onto the dSPB in the bud, thereby preventing spurious inhibition of the MEN.
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