Nuclear Pores Protect Genome Integrity by Assembling a Premitotic and Mad1-Dependent Anaphase Inhibitor Veronica Rodriguez-Bravo,1 John Maciejowski,1 Jennifer Corona,1 Ha˚ kon Kirkeby Buch,2,5 Philippe Collin,3 Masato T. Kanemaki,4 Jagesh V. Shah,5 and Prasad V. Jallepalli1,* 1Molecular Biology Program and Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA 2Swiss Federal Institute of Technology Zu¨ rich, CH-8093 Zu¨ rich, Switzerland 3The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge CB2 1QN, UK 4Center for Frontier Research, National Institute of Genetics, ROIS, and Department of Genetics, SOKENDAI, Yata 1111, Mishima, Shizuoka 411-8540, Japan 5Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2014.01.010 SUMMARY also implicates NPCs in modes of regulation that are distinct from nuclear transport. For example, some actively transcribed The spindle assembly checkpoint (SAC) delays genes in yeast are tethered to NPCs via bridging complexes anaphase until all chromosomes are bioriented on that also recruit transcription factors and mRNA-processing the mitotic spindle. Under current models, unat- enzymes, thereby enhancing gene expression at multiple levels tached kinetochores transduce the SAC by cata- (Dieppois and Stutz, 2010; Strambio-De-Castillia et al., 2010). lyzing the intramitotic production of a diffusible By comparison, metazoan Nups stimulate transcription by inter- inhibitor of APC/CCdc20 (the anaphase-promoting acting with target loci within the nuclear interior (Capelson and Hetzer, 2009). Further redistribution and repurposing occur complex/cyclosome and its coactivator Cdc20, a during mitosis, as NPC disassembly and nuclear envelope large ubiquitin ligase). Here we show that nuclear breakdown (NEBD) enable the Nup107-160 complex, Nup358/ pore complexes (NPCs) in interphase cells also func- RanBP2, and the exportin Crm1 to relocalize at kinetochores, tion as scaffolds for anaphase-inhibitory signaling. where these proteins regulate microtubule dynamics in conjunc- This role is mediated by Mad1-Mad2 complexes tion with RanGTP (Arnaoutov et al., 2005; Joseph et al., 2004; tethered to the nuclear basket, which activate solu- Zuccolo et al., 2007). ble Mad2 as a binding partner and inhibitor of Another example of NPC-to-kinetochore migration involves Cdc20 in the cytoplasm. Displacing Mad1-Mad2 the Mad1-Mad2 complex. This heterodimer acts as the terminal from nuclear pores accelerated anaphase onset, pre- transducer of the spindle assembly checkpoint (SAC) that delays vented effective correction of merotelic errors, and anaphase until all kinetochores are bound by microtubules increased the threshold of kinetochore-dependent (Foley and Kapoor, 2013; Musacchio and Salmon, 2007). During interphase, Mad1 and Mad2 are docked at the nucleoplasmic signaling needed to halt mitosis in response to side of the NPC, principally through interactions with a con- spindle poisons. A heterologous Mad1-NPC tether served family of coiled-coil proteins (Tpr in vertebrates, Megator restored Cdc20 inhibitor production and normal in flies, and Mlp1/2 in yeast) that make up the nuclear basket M phase control. We conclude that nuclear pores (Campbell et al., 2001; Lee et al., 2008; Lince-Faria et al., and kinetochores both emit ‘‘wait anaphase’’ signals 2009; Scott et al., 2005). This arrangement persists until NEBD, that preserve genome integrity. when the Mad1-Mad2 complex is recruited to unattached kinet- ochores by upstream components of the SAC, including the Mps1, Aurora B, and Bub1 kinases, and the Rod-Zw10-Zwilch INTRODUCTION complex (Foley and Kapoor, 2013; Musacchio and Salmon, 2007). Compelling evidence indicates that Mad1 binding shifts A defining feature of all eukaryotes is the nuclear envelope (NE), Mad2 from its ‘‘open’’ (O or N1) to ‘‘closed’’ (C or N2) conforma- which divides the cell into spatially and functionally distinct com- tion, which not only stabilizes the heterodimer but also endows it partments. Macromolecular traffic across the NE is mediated by with prion-like activity, whereby it can induce a similar structural nuclear pore complexes (NPCs), large transmembrane cylinders change in soluble O-Mad2 (Musacchio and Salmon, 2007; Yu, formed from 30 nucleoporins (Nups), and soluble transport re- 2006). As C-Mad2, this pool can bind Cdc20, a key activator of ceptors that shuttle cargoes in response to the Ran GTPase the anaphase-promoting complex or cyclosome (APC/C), a large (Hetzer and Wente, 2009; Stewart, 2007). Mounting evidence ubiquitin ligase (Pines, 2011). In conjunction with a second Cell 156, 1017–1031, February 27, 2014 ª2014 Elsevier Inc. 1017 Figure 1. Gene Deletion Reveals Mad1’s Dual Roles in Mitotic Timing and Check- point Enforcement (A) RPE cells in which one or both MAD1L1 alleles had been targeted with AAV vectors (see Fig- ure S1) were infected with AdCre and sampled for 6 days thereafter. (B) Cells in (A) were treated with nocodazole and MG132 for 90 min. Maximum-intensity projections and magnified views of kinetochores (insets) are shown. Mad1 and Mad2 signals were normalized against CREST. Scale bar, 5 mm. (C) Cells expressing H2B-GFP were traced during an unperturbed mitosis. Time 0 denotes NEBD. Arrowheads highlight lagging chromatids and micronuclei. (D) The interval from NEBD to anaphase (left plot) or furrow ingression (right plot) was deter- mined from time-lapse recordings. See Movies S1 and S2. (E and F) HCT116 cells were modified at the MAD1L1 locus, infected with AdCre, and analyzed as above. Where indicated, Mps1-IN-1 or re- versine was added. Data sets in (D) and (F) were compared by Student’s t test. controversial (Lee et al., 2008; Lince- Faria et al., 2009; Schweizer et al., 2013). We used genetic and computational methods to investigate the functions and regulation of human Mad1. Here we show that NPC tethering allows the Mad1-Mad2 dimer to initiate MCC assembly before cells reach mitosis (Su- dakin et al., 2001). By proactively inhibit- ing APC/CCdc20, the NPC-derived ‘‘wait anaphase’’ signal buffers its intramitotic counterpart, which is regulated by kineto- chore-microtubule attachment and es- tablished after NEBD. Together the two Cdc20 inhibitor, BubR1, and its cofactor Bub3, C-Mad2 and systems make the SAC more sensitive and robust and facilitate Cdc20 form one or more mitotic checkpoint complexes the correction of mitotic errors that are invisible to the SAC. (MCCs; Fang, 2002; Sudakin et al., 2001; Tang et al., 2001) Collectively our results define a new role of the interphase NE that inhibit APC/C-mediated proteolysis of securin and cyclin in signal transduction and genome maintenance that outlasts B, thereby delaying sister-chromatid separation and mitotic its disassembly. exit (Foley and Kapoor, 2013; Musacchio and Salmon, 2007). In contrast, the roles of Mad1 and Mad2 at interphase NPCs RESULTS remain ill-defined. One hypothesis, namely that one or both SAC mediators modulate traffic across the NE, is supported by Mitotic Timing and Checkpoint Defects in MAD1L1-Null the finding that yeast Mad1 cycles between kinetochores and Cells NPCs to inhibit Kap121-mediated nuclear import during this Because Mad1 RNAi often fails to elicit a SAC defect (Fava et al., organism’s closed mitosis (Cairo et al., 2013). However, higher 2011), we used adeno-associated virus (AAV)-mediated gene organisms synchronize both NPC disassembly and kinetochore targeting to modify the MAD1L1 locus in human retinal pigment assembly with the start of M phase, eliminating opportunities for epithelial cells, such that exon 12 was either flanked by loxP sites equivalent crosstalk (Cheeseman and Desai, 2008; Hetzer and or deleted outright (Figures S1A and S1B available online). Next, Wente, 2009). Although the Nups responsible for recruiting MAD1L1flox/D cells or controls were infected with an adenovirus Mad1 and Mad2 to the NE have been suggested to support expressing Cre recombinase. Over the next 3 to 6 days, Mad1 SAC activity in metazoans, how this occurs is still unclear and was depleted (Figure 1A), which in turn abolished Mad2’s 1018 Cell 156, 1017–1031, February 27, 2014 ª2014 Elsevier Inc. targeting to kinetochores (Figure 1B) and mitotic arrest in mura et al., 2009) and wild-type or aid (auxin-inducible degron)- response to spindle poisons like nocodazole (Figure S1C). tagged Mad1 (Figure 2E). Whereas aid-Mad1 was destabilized Having validated the penetrance of our system, we asked how by auxins, wild-type Mad1 was immune (Figures 2F and 2G). Mad1 contributes to progression through an otherwise unper- Crucially, a brief pulse of aid-Mad1 destruction was enough to turbed mitosis. Previous studies have defined two modes liberate Cdc20 from Mad2 in interphase extracts (Figures 2G through which anaphase can be delayed by the SAC network. and 2H), demonstrating that Mad1 is continuously required for The first and more familiar pathway uses kinetochores to activate MCC assembly in interphase cells. Mad2 as a Cdc20-binding partner and inhibitor (Foley and Kapoor, 2013; Musacchio and Salmon, 2007). However, a large Mad1-Mad2 Dimers Are Required for Interphase MCC fraction of Mad2 and BubR1 are already bound to Cdc20 during Assembly but Not Nuclear Transport interphase and appear to define the minimal length of M phase In light of Mad1’s association with nuclear pores, which have independently of kinetochores (Maciejowski et al., 2010; Malur- both transport-dependent and -independent roles in other eanu et al., 2009; Meraldi et al., 2004; Sudakin et al., 2001). processes (Capelson and Hetzer, 2009; Strambio-De-Castillia Notably, this interphase-specific inhibitor or ‘‘mitotic timer’’ still et al., 2010), we considered two different explanations for these requires Mps1 (Maciejowski et al., 2010) but supposedly not results. First, Mad1 might control the transport of specific MCC Mad1 (Meraldi et al., 2004), raising the question of where and subunits or regulators in humans, analogous to its role in inhibit- how Mad2 is activated during interphase.