Molecular mechanism of dynein recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly

JCB JCB: Article THE JOURNAL OF CELL BIOLOGY 943 The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 4 943–960 https://doi.org/10.1083/jcb.201610108 Introduction Chromosome segregation during cell division requires attachments between spindle microtubules and kinetochores, multiprotein complexes that assemble on each sister chromatid. The microtubule-based motor cytoplasmic dynein 1 (dynein) localizes to the outermost layer of the kinetochore, the fibrous corona, which expands in early prometaphase when kinetochores are not yet occupied by microtubules (Hoffman et al., 2001; Wynne and Funabiki, 2016). Corona expansion enlarges the kinetochore surface available for initial lateral microtubule capture by dynein, which drives transient poleward motion of chromosomes and accelerates the formation of stable endcoupled attachments by the Ndc80 complex (Rieder and Alexander, 1990; Yang et al., 2007; Vorozhko et al., 2008; Barisic et al., 2014; Cheerambathur and Desai, 2014; Magidson et al., 2015; Wynne and Funabiki, 2015). Corona expansion also facilitates amplification of the diffusible “wait anaphase” signal by the spindle assembly checkpoint (SAC) that prevents premature sister chromatid separation (Musacchio, 2015; Wynne and Funabiki, 2015). After kinetochore microtubule attachment, dynein contributes to corona disassembly and SAC silencing by transporting SAC components from kinetochores to spindle poles as part of a motor–cargo complex (Howell et al., 2001; Wojcik et al., 2001). Dynein recruitment to kinetochores is dependent on the three-subunit Rod–Zw10–Zwilch (RZZ) complex and the coiled-coil protein Spindly (Starr et al., 1998; Griffis et al., 2007; Gassmann et al., 2008; Yamamoto et al., 2008; Chan et al., 2009; Barisic et al., 2010), but the molecular mechanism remains elusive. RZZ is also required for recruitment of Mad1/ Mad2, whose presence at kinetochores is essential for SAC activation (Buffin et al., 2005; Karess, 2005; Kops et al., 2005; Essex et al., 2009). RZZ subunits are interdependent for kinetochore localization, consistent with the observation that their inhibition causes identical defects (Smith et al., 1985; Karess and Glover, 1989; Williams and Goldberg, 1994; Starr et al., 1997; Scaërou et al., 1999, 2001; Chan et al., 2000; Williams et al., 2003; Gassmann et al., 2008; Wainman et al., 2012). Anaphase chromosome bridges are a hallmark of RZZ inhibition in Drosophila melanogaster and Caenorhabditis elegans, and the The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod–Zw10–Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod’s N-terminal β-propeller and the associated Zwilch subunit bind Spindly’s C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod β-propeller–Zwilch complex in vitro. Spindly’s N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein–dynactin–Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein–dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo. Molecular mechanism of dynein recruitment to kinetochores by the Rod–Zw10–Zwilch complex and Spindly JoséB.Gama,1,2* CláudiaPereira,1,2* PatríciaA.Simões,1,2* RicardoCelestino,1,2 RitaM.Reis,1,2 DanielJ.Barbosa,1,2 HelenaR.Pires,1,2 CátiaCarvalho,1,2 JoãoAmorim,1,2 AnaX.Carvalho,1,2 DhanyaK.Cheerambathur,3,4 and RetoGassmann1,2 1Instituto de Biologia Molecular e Celular and 2Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal 3Ludwig Institute for Cancer Research and 4Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 © 2017 Gama et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http ://www .rupress .org /terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /). *J.B.Gama, C.Pereira, and P.A.Simões contributed equally to this paper. Correspondence to Reto Gassmann: [email protected] Abbreviations used: ACA, anticentromere antibody; dsRNA, double-stranded RNA; IC, intermediate chain; LIC, light intermediate chain; MBP, maltosebinding protein; RZZ, Rod–Zw10–Zwilch; SAC, spindle assembly checkpoint; TEV, tobacco etch virus. on January 19, 2018jcb.rupress.orgDownloaded from http://doi.org/10.1083/jcb.201610108 Supplemental material can be found at: JCB • Volume 216 • NumBer 4 • 2017944 resulting aneuploidy is lethal, underscoring the importance of RZZ for the fidelity of chromosome segregation. Spindly acts downstream of RZZ as a kinetochorespecific adaptor for dynein, and in C.elegans it is also required for kinetochore recruitment of Mad1/Mad2 (Gassmann et al., 2008; Yamamoto et al., 2008). Whether RZZ directly binds to Spindly is unclear, nor is it known which RZZ subunits mediate Spindly recruitment. Like other adaptor proteins, Spindly forms a ternary complex with dynein and its cofactor dynactin, which facilitates processive movement of the motor in vitro (Splinter et al., 2012; McKenney et al., 2014). Spindly proteins contain a highly conserved motif in their N-terminal coiled-coil region, point mutations in which abrogate dynein–dynactin recruitment in vivo (Gassmann et al., 2010; Cheerambathur et al., 2013). The molecular basis of Spindly’s interaction with dynein– dynactin and the role of the Spindly motif are not known. In this study, we describe protein–protein interactions that link RZZ to Spindly and Spindly to dynein and dynactin. We demonstrate the functional relevance of these interactions with engineered mutants biochemically and in vivo using the C.elegans early embryo and cultured human cells, thus providing a molecular view of how dynein is recruited to kinetochores. Our results suggest that the mechanism used by Spindly to engage dynein and dynactin is also relevant for how the motor interacts with cargo adaptors in the context of intracellular transport. Results ROD-1 and Zw10CZW-1 can target to kinetochores without ZwilchZWL-1 To gain insight into the mechanisms of kinetochore dynein recruitment by the RZZ complex and Spindly (the kinetochore dynein module; Fig.1A), we asked how the three subunits of RZZ contribute to the function of the complex. We used the C.elegans early embryo, in which previous work had delineated the contribution of the kinetochore dynein module to chromosome segregation (Gassmann et al., 2008; Cheerambathur et al., 2013). We first defined the basic architecture of C.elegans RZZ using yeast two-hybrid mapping (Fig. S1, A–C). The predicted N-terminal β-propeller domain of ROD-1 (residues 1–373) was necessary and sufficient for the interaction with ZwilchZWL-1, and the Zw10CZW-1 binding region of ROD-1 mapped to residues 500–1,203 within ROD-1’s α-solenoid region, which corresponds to the Sec39 domain. Accordingly, residues 1–1,203 interacted with both ZwilchZWL-1 and Zw10CZW-1. No interaction was detected between ZwilchZWL-1 and Zw10CZW-1.Thus, ZwilchZWL-1 and Zw10CZW-1 bind independently to distinct domains within ROD-1’s N-terminal half (Fig.1B). Next, we generated animals expressing mCherry-tagged ROD-1, ZwilchZWL-1, and Zw10CZW-1 to examine the localization dependency of RZZ subunits (Fig.1C). The transgenes were made resistant to RNAi by codon reshuffling and integrated in single copy at a defined chromosomal locus (Fig. S1 D; Frøkjær-Jensen et al., 2012). Consistent with prior work (Gassmann et al., 2008), kinetochore localization of ZwilchZWL-1::mCherry and mCherry::Zw10CZW-1 was lost after depletion of ROD-1 (Fig. 1, D and E), and kinetochore localization of mCherry::ROD-1 depended on Zw10CZW-1 (Fig. 1 F). Immunoblotting showed that mCherry::ROD-1 loss from kinetochores after Zw10CZW-1 depletion could be attributed to destabilization of mCherry::ROD-1 (Fig.1H). In contrast, depletion of ZwilchZWL-1 did not destabilize mCherry::ROD-1, facilitating localization dependency analysis. Depletion of endogenous ROD-1 increased kinetochore mCherry::ROD-1 levels 2.4-fold (Fig.1G), presumably because mCherry::ROD-1 did not have to compete with endogenous ROD-1 for kinetochore targeting. Remarkably, and contrary to expectations, we observed a similar increase in kinetochore mCherry::ROD-1 levels after depletion of ZwilchZWL-1 (2.8-fold; Fig.1, F and G). This suggested that endogenous ROD-1, but not mCherry::ROD-1, was destabilized in the absence of ZwilchZWL-1.Immunofluorescence with antibodies against ROD-1 confirmed that depletion of ZwilchZWL-1 abolished endogenous ROD-1 localization to kinetochores (Fig.1I), whereas robust kinetochore ROD-1 signal was detected in embryos expressing mCherry::ROD-1. Importantly, kinetochore-localized mCherry::ROD-1 recruited GFP::Zw10CZW-1 in ZwilchZWL-1-depleted embryos (Fig.1J), consistent with the independent binding of Zw10CZW-1 and ZwilchZWL-1 to ROD-1 observed in the yeast two-hybrid assay. These results indicate that ZwilchZWL-1 does not make a direct contribution to kinetochore targeting of ROD-1 and Zw10CZW-1. Instead, the displacement of ROD-1 and Zw10CZW-1 from kinetochores in ZwilchZWL-1-depleted embryos is likely an indirect consequence of ROD-1 destabilization. Fusing mCherry to ROD1’s β-propeller domain, where ZwilchZWL-1 binds (Fig. S1, A–C), may stabilize ROD-1 when ZwilchZWL-1 is absent, thus facilitating mCherry::ROD-1 targeting to kinetochores along with Zw10CZW-1 (Fig.1K). ZwilchZWL-1 is required for kinetochore recruitment of SpindlySPDL-1 We next asked whether the kinetochore-localized mCherry::ROD-1–Zw10CZW-1 subcomplex was sufficient to support the recruitment of SpindlySPDL-1.Depletion of endogenous ROD-1 confirmed that mCherry::ROD-1 could recruit GFP::SpindlySPDL-1 to kinetochores (Fig. 2 A). In contrast, depletion of ZwilchZWL-1 displaced GFP::SpindlySPDL-1 from kinetochores despite kinetochore-localized mCherry::ROD-1. Importantly, immunoblotting demonstrated that depletion of ZwilchZWL-1 did not affect total SpindlySPDL-1 levels (Fig. 1 H). These results directly implicated ZwilchZWL-1 in the kinetochore targeting of SpindlySPDL-1. Identification of conserved ZwilchZWL-1 residues required for SpindlySPDL-1 recruitment to kinetochores We sought to identify structural features in ZwilchZWL-1 required for kinetochore targeting of SpindlySPDL-1.Sequence alignments showed that E433 and E437 of ZwilchZWL-1 occupied the same positions in the αJ helix of domain 2 as human E422 and D426, which had previously been predicted to participate in a protein–protein interaction based on their conservation and surface-exposed position in the structure (Fig.2B; Çivril et al., 2010). Mutating both E433 and E437 to alanine (E/A) did not affect the yeast two-hybrid interaction between ZwilchZWL-1 and full-length ROD-1 (Fig. 2 C). Furthermore, when expressed recombinantly in insect cells, both wild-type ZwilchZWL-1 and the E/A mutant copurified with 6×His-tagged ROD-1 β-propeller (residues 1–372), and the complexes had identical elution profiles in size exclusion chromatography (Fig. S2, A and B). Thus, E433 and E437 of ZwilchZWL-1 are unlikely to be involved in RZZ complex formation. We generated animals expressing RNAi-resistant ZwilchZWL-1(E/A)::mCherry from the same on January 19, 2018jcb.rupress.orgDownloaded from Dynein recruitment to kinetochores • Gama et al. 945 Figure 1. ROD-1 and Zw10CZW-1 can target to kinetochores independently of ZwilchZWL-1.(A) Cartoon depicting localization dependencies of kinetochore dynein module components. (B) Summary of interactions between C.elegans RZZ subunits, based on yeast two-hybrid mapping (Fig. S1, A–C). Note that the intact RZZ complex likely exists as a dimer of the ROD-1–Zw10CZW-1–ZwilchZWL-1 trimer, based on hydrodynamic analysis of the human and D.melanogaster complexes (Williams et al., 2003; Çivril et al., 2010). (C) Cartoon of the one-cell embryo spindle region shown in subsequent panels. (D–F) Localization dependency analysis of mCherry-tagged RZZ subunits. Still images are from time-lapse sequences. (G) Quantification of mCherry::ROD-1 levels at kinetochores in the conditions shown in D–F.Circles correspond to measurements in individual one-cell embryos in metaphase. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***, P < 0.0001 compared with no RNAi control; ns, P > 0.05). (H) Immunoblots showing that depletion of Zw10CZW-1 but not ZwilchZWL-1 lowers mCherry::ROD-1 levels. α-Tubulin served as the loading control. (I) Immunofluorescence images of metaphase kinetochores stained for ROD-1.Endogenous ROD-1 is missing from kinetochores in ZwilchZWL-1depleted embryos, whereas mCherry::ROD-1 localizes under the same conditions. (J) Stills from a time-lapse sequence showing that mCherry::ROD-1 supports kinetochore localization of GFP::Zw10CZW-1 in the absence of ZwilchZWL-1.Bars: (D–F and J) 5 µm; (I) 2µm. (K) Cartoon summary of the data shown in C–J. on January 19, 2018jcb.rupress.orgDownloaded from JCB • Volume 216 • NumBer 4 • 2017946 on January 19, 2018jcb.rupress.orgDownloaded from Dynein recruitment to kinetochores • Gama et al. 947 chromosomal locus as our animals expressing the wild-type transgene. In contrast to wild-type ZwilchZWL-1::mCherry, the E/A mutant caused high embryonic lethality when endogenous ZwilchZWL-1 was depleted, despite identical expression levels (Fig.2, D and E). No decrease in embryonic viability was observed in the presence of endogenous ZwilchZWL-1, demonstrating that the E/A mutant did not act as a dominant negative. ZwilchZWL-1(E/A)::mCherry supported kinetochore localization of endogenous ROD-1 and Zw10CZW-1 (Fig.2, F and G), and both wild-type ZwilchZWL-1::mCherry and the E/A mutant localized equally well to kinetochores (Fig.2, H and I). Wildtype ZwilchZWL-1::mCherry also recruited GFP::SpindlySPDL-1 to kinetochores (Fig. 2, H and I; and Video 1). In contrast, GFP::SpindlySPDL-1 was delocalized from kinetochores harboring the Zwilch E/A mutant. The Zwilch E/A mutant also delocalized dynein, dynactin, and Mad2MDF-2 from kinetochores and failed to activate the SAC, as predicted by the absence of SpindlySPDL-1 from kinetochores (Fig. S2, C–F). Depletion of SpindlySPDL-1 resulted in severe chromosome segregation defects because without dynein at kinetochores, RZZ inhibits the microtubule-binding activity of NDC-80 (Fig.2J; Gassmann et al., 2008; Cheerambathur et al., 2013). In contrast, depletion of RZZ subunits resulted in milder defects that reflect a loss of kinetochore dynein without NDC-80 inhibition. Visualization of chromosome dynamics showed that the ZwilchZWL-1 E/A mutant caused defects in chromosome segregation that were significantly more severe than depleting ZwilchZWL-1 on its own and resembled those of SpindlySPDL-1 depletions (Fig.2J and Video2). This was also evident by quantifying the kinetics of spindle pole separation, which is a readout for the formation of end-coupled kinetochore–microtubule attachments capable of resisting tension (Fig. S2, G and H). Collectively, these results demonstrate that mutating residues E433 and E437 of ZwilchZWL-1 to alanine selectively displaces SpindlySPDL-1 from kinetochores without perturbing RZZ localization. The C-terminal domain of SpindlySPDL-1 binds to ZwilchZWL-1 and the ROD-1 β-propeller Next, we asked whether RZZ binds directly to SpindlySPDL-1.Initial attempts failed to reveal an interaction between ZwilchZWL-1 and SpindlySPDL-1 when both components were purified (unpublished data). We therefore performed pull-downs from insect cell lysates containing different recombinant versions of C. elegans RZZ (Fig. 3 A). Purified GST::SpindlySPDL-1 (Fig. 3 B) pulled down full-length RZZ, ROD-11–1,203ZZ, ROD-11–372–ZwilchZWL-1, and ZwilchZWL-1 by itself from corresponding lysates, and replacing wild-type ZwilchZWL-1 with the E/A mutant diminished these interactions (Fig. 3, C and D; and Fig. S3). We conclude that SpindlySPDL-1 interacts with RZZ through the ZwilchZWL-1 subunit and that the interaction requires residues E433 and/or E437 in ZwilchZWL-1.Interestingly, GST::SpindlySPDL-1 also pulled down ROD-11–372 by itself from lysate (Fig.3, E and F), suggesting that SpindlySPDL-1 interacts with RZZ through both ZwilchZWL-1 and the ROD-1 β-propeller. To narrow down the binding region in SpindlySPDL-1, we purified its N-terminal coiled-coil region (residues 1–361) and the C-terminal domain (residues 362–479; Fig. 3 B). GST pulldowns from lysates revealed that the SpindlySPDL-1 C-terminal domain contains the binding sites for both ZwilchZWL-1 and ROD-11–372 (Fig.3F). Although GST::SpindlySPDL-1 pulled down ROD-11–372 by itself from lysates (Fig.3F), GST::SpindlySPDL-1 did not pull down full-length ROD-1 from lysates when ZwilchZWL-1 E/A was present (Figs. 3 C and S3 A). A possible explanation is that free ROD-1 β-propeller has higher affinity for SpindlySPDL-1 than ROD-1 β-propeller bound to ZwilchZWL-1, such that the SpindlySPDL-1–ZwilchZWL-1 interaction becomes essential for the binding of SpindlySPDL-1 to RZZ. The residual amounts of ROD11–1,203 and ROD-11–372 pulled down by GST::SpindlySPDL-1 in the presence of ZwilchZWL-1 E/A (Fig.3, C and D) may therefore represent a pool of free ROD-1 that is not bound to ZwilchZWL-1 E/A in the lysate (note that our in vivo data in Fig.1 suggest that full-length ROD-1 is unstable when not bound to ZwilchZWL-1). To explore this idea further, we repeated the pull-downs after purifying ROD-11–372 on its own and in complex with ZwilchZWL-1 (Fig.3G). GST::SpindlySPDL-1 pulled down free ROD-11–372 and ROD-11–372 bound to wild-type ZwilchZWL-1, but not ROD-11–372 bound to the ZwilchZWL-1 E/A mutant (Fig.3, H and I). Thus, although SpindlySPDL-1 binds free ROD-11–372, the SpindlySPDL-1– ROD-11–372 interaction is not sufficient to mediate binding of SpindlySPDL-1 to the ROD-11–372–ZwilchZWL-1 E/A complex. This result agrees with the observation that the ZwilchZWL-1 E/A mutant abrogates kinetochore recruitment of SpindlySPDL-1 despite kinetochore-localized ROD-1 (Fig.2, F and H). Collectively, biochemical and in vivo analysis suggests that RZZ recruits SpindlySPDL-1 to kinetochores through a bipartite interaction between its ZwilchZWL-1–ROD-1 β-propeller module and the SpindlySPDL-1 C-terminal domain (Fig.3J). Human Rod without its β-propeller targets Zw10 to kinetochores but fails to recruit Zwilch and Spindly To assess whether the function of the Zwilch–Rod β-propeller module is conserved in human cells, we characterized human Figure 2. ZwilchZWL-1 residues required for SpindlySPDL-1 recruitment to kinetochores. (A) Stills from time-lapse sequences showing that kinetochore-localized mCherry::ROD-1 does not support GFP::SpindlySPDL-1 recruitment in the absence of ZwilchZWL-1.(B) Cartoon of domain architecture and sequence alignment of Zwilch homologues. Residues E433 and E437 were both mutated to alanine (E/A). (C) Yeast two-hybrid assay showing that the ZwilchZWL-1 E/A mutant interacts with full-length ROD-1.Cells containing bait and prey plasmids grow on -Leu/-Trp plates, whereas -Leu/-Trp/-His plates select for bait–prey interaction. (D) Immunoblots of C.elegans adult worms expressing transgene-encoded ZwilchZWL-1::mCherry wild-type (WT) or the E/A mutant. The asterisk denotes a cross-reacting protein band of similar size as ZwilchZWL-1::mCherry. α-Tubulin served as the loading control. (E) Embryonic viability assay. More than 300 embryos from 12 or more mothers were counted for each condition. (F and G) Still images from a time-lapse sequence showing robust kinetochore localization of ROD-1 and 3×FLAG::Zw10CZW-1 in the ZwilchZWL-1 E/A mutant. (H) Selected frames from a time-lapse sequence showing that ZwilchZWL-1::mCherry E/A fails to recruit GFP::SpindlySPDL-1 (see Video1). (I) Quantification of ZwilchZWL-1::mCherry and GFP::SpindlySPDL-1 levels on metaphase kinetochores in the conditions shown in H.Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***, P < 0.0001; ns, P > 0.05). (J, left) Cartoon summarizing the inhibitory cross-talk between the kinetochore dynein module and NDC-80. (Right) Selected frames from time-lapse sequences of the first embryonic division, demonstrating that the chromosome segregation defects of ZwilchZWL-1::mCherry E/A resemble those of SpindlySPDL-1 depletion (see Video2). The frequencies of anaphase chromatin bridges are indicated in the percentages and absolute numbers of embryos in parentheses. Time is relative to the onset of sister chromatid separation. Bars: (A, H, and J) 5 µm; (F and G) 2 µm. on January 19, 2018jcb.rupress.orgDownloaded from JCB • Volume 216 • NumBer 4 • 2017948 Figure 3. SpindlySPDL-1 binds ZwilchZWL-1 and the ROD-1 β-propeller. (A) Lysates of insect Sf21 cells coinfected with viruses to express full-length (FL) ROD11–2,177ZZ, ROD-11–1,203ZZ, ROD-11–372–ZwilchZWL-1, or ZwilchZWL-1 alone. ROD-1 and ZwilchZWL-1 were tagged with 6×His for detection on immunoblots. (B) Coomassie-stained protein gels showing purified recombinant GST::SpindlySPDL-1 used in pull-downs. (C and D) GST pull-downs from the lysates in A with purified GST::SpindlySPDL-1 from B.Protein fractions bound to beads were analyzed by immunoblotting using anti-6×His antibody. The same membranes were then reprobed with anti-GST antibody. (E) Lysates of insect Sf21 cells infected with viruses encoding for the ROD-1 β-propeller (residues 1–372) or ZwilchZWL-1, both tagged with 6×His. (F) Immunoblots of GST pull-downs from the lysates in E with purified GST::SpindlySPDL-1 (see A). (G) Coomassiestained protein gels showing purified ROD-1 β-propeller alone and in complex with wild-type (WT) ZwilchZWL-1 or the E/A mutant. (H and I) Immunoblots of GST pull-downs using the purified proteins in B and G.(J) Cartoon showing the bipartite interaction between RZZ and the C-terminal domain (CTD) of SpindlySPDL-1.Molecular mass is indicated in kilodaltons. on January 19, 2018jcb.rupress.orgDownloaded from Dynein recruitment to kinetochores • Gama et al. 949 Rod lacking the β-propeller domain (Δ1–375). We integrated expression constructs for full-length GFP::hRod and the Δ1–375 mutant into a single genomic locus in HeLa cells. Transgenes were resistant to an siRNA oligonucleotide that efficiently depleted endogenous hRod on immunoblots and delocalized Spindly, Mad1, and Zw10 from kinetochores by immunofluorescence (Fig. 4, A–D). Full-length GFP::hRod localized to kinetochores in cells treated with hRod siRNA and nocodazole (to avoid effects on kinetochore levels caused by microtubule binding) and supported kinetochore recruitment of Zwilch, Zw10, Spindly, and Mad1 (Fig. 4, E–K). GFP::hRod(Δ1–375) localized robustly to kinetochores in hRoddepleted cells (Fig. 4 E). However, the crescent morphology typically adopted by kinetochores after nocodazole treatment was distinctly missing (Fig. 4 E, blowups). This suggests that GFP::hRod(Δ1–375)-containing kinetochores were unable to undergo corona expansion in response to microtubule depolymerization. Accordingly, kinetochore levels of GFP::hRod(Δ1–375) in nocodazole-treated cells were reduced by 37% relative to full-length GFP::hRod (Fig.4F). Despite this difference, GFP::hRod(Δ1–375) was as efficient in recruiting Zw10 to kinetochores as full-length GFP::hRod (Fig.4, G and K). In contrast, GFP::hRod(Δ1–375) failed to recruit Zwilch or Spindly to kinetochores (Fig.4, H, I, and K). We also observed a modest reduction of Mad1 levels in GFP::hRod(Δ1–375) relative to full-length GFP::hRod (Fig. 4, J and K). In prometaphase cells without nocodazole treatment, full-length GFP::hRod was enriched at spindle poles along with Zw10, Zwilch, Spindly, and Mad1 (Fig.4, L–O). Spindle pole enrichment was missing in cells expressing GFP::hRod(Δ1–375). This is consistent with the loss of Spindly from kinetochores, which abrogates dynein-mediated poleward transport of corona components (Gassmann et al., 2010). We conclude that the role of the Rod β-propeller–Zwilch module in Spindly recruitment is conserved in human cells. Furthermore, removing the βpropeller from hRod impairs corona expansion in response to microtubule depolymerization. Spindly binds dynein light intermediate chain (LIC) and dynactin’s pointed-end complex through two conserved motifs We next addressed how Spindly recruits dynein and dynactin. Spindly forms a ternary complex with dynein–dynactin in vitro, but molecular details are not known (McKenney et al., 2014). The C-terminal region of dynein LIC interacts with multiple adaptors for membrane cargo (Fig.5A; Schroeder et al., 2014; Schroeder and Vale, 2016). We therefore asked whether LIC also bound Spindly using purified human proteins. A GST fusion of full-length LIC1 (residues 1–523) or its C-terminal region (residues 388–523) pulled down Spindly’s N-terminal coiled-coil region (residues 2–359) but not its C-terminal region (residues 360–605; Fig.5, C and D). Sequence alignments revealed a region in Spindly’s first coiled-coil segment that is conserved in multiple dynein adaptors, including BICD2 (Figs. 5 B and S4 A; Schlager et al., 2014; Hoogenraad and Akhmanova, 2016). Mutating the region’s two conserved alanines to valines in BICD2 inhibits complex formation with dynein–dynactin (Schlager et al., 2014), and the mutation A40V in D.melanogaster BICD causes a hypomorphic loss-of-function phenotype (Oh et al., 2000). We introduced the two alanine-to-valine (A/V) mutations into Spindly2–359 and BICD22–422 and found that both mutant proteins failed to bind LIC1 in GST pull-downs (Fig.5, C and D; and Fig. S4, B–D). Furthermore, BICD22–422 displaced Spindly2–359 from GST::LIC1 in titration experiments (Fig. S4 E). We conclude that Spindly and BICD2 engage with the C-terminal region of LIC1 through a similar mechanism that involves a conserved region in their N-terminal coiled-coil segment, hereafter referred to as the CC1 box. We previously showed that point mutations in the Spindly motif, which is located more C-terminally to the CC1 box in Spindly’s coiled-coil region (Figs. 5 F and S4 A), abrogate dynein and dynactin recruitment to kinetochores in human cells (S256A or F258A) and C.elegans (F199A; Gassmann et al., 2010; Cheerambathur et al., 2013). We found that binding of Spindly to LIC1 was not affected by the F258A mutation (Fig. 5, C and D), implying that the Spindly motif mediated an additional distinct interaction with either dynein or dynactin. Structural work on the dynein–dynactin–BICD2 complex showed that the coiled-coil formed by BICD2’s N-terminal 270 residues binds along dynactin’s Arp1 filament, with the C-terminal end of the coiled-coil exiting at the filament’s pointed end (Urnavicius et al., 2015). If Spindly’s coiled-coil region also binds along the Arp1 filament, the Spindly motif around residue 258 would be predicted to reside near the filament’s pointed end, which is capped by a complex consisting of Arp11, p62, p27, and p25 (Fig.5E). We found that Streptagged Spindly2–359 pulled down purified recombinant pointed-end complex and that the association was diminished in the Spindly F258A mutant but not in the A/V mutant (Fig.5, G and H). Thus, Spindly binds the pointed-end complex of dynactin in vitro, and this interaction involves a motif required for dynein– dynactin recruitment in vivo. We next tested whether the mutations in the CC1 box and the Spindly motif affected binding of Spindly to intact dynein and dynactin in porcine brain lysate. Strep-tagged Spindly2–359 pulled down dynein and dynactin from lysate, as revealed by immunoblotting for dynactin p150 and dynein intermediate chain (IC; Fig. 5 I). In contrast, both Spindly2–359 A/V and F258A pulled down significantly less p150 and IC. Thus, binding of Spindly to LIC1 and the dynactin pointed end is required for the assembly of a stable ternary complex between dynein, dynactin, and Spindly. A null allele of p27 dnc-6 prevents dynein– dynactin recruitment to kinetochores Our results suggested that dynactin’s pointed-end complex has a key role in recruiting dynein–dynactin to kinetochores through Spindly. To directly test this prediction, we analyzed the loss-of-function phenotype of C.elegans pointed-end complex subunits in the mitotic one-cell embryo. RNAi-mediated depletion of Arp-11 and p62DNC-4 mimicked the phenotype of p150DNC-1 depletion, characterized by failure of centrosome separation and defective pronuclear migration (Fig. S5 A). This “dynactin-null” phenotype is consistent with a role for Arp-11 and p62DNC-4 in Arp-1 filament stabilization, as described for the mammalian proteins (Yeh et al., 2012). Similar, if less severe, defects in pronuclear migration were also evident after depletion of p25DNC-5.In contrast, depletion of p27DNC-6 had no adverse effects on centrosome separation, pronuclear migration, or spindle positioning (Fig. S5 A). Instead, we observed a specific defect in chromosome segregation (Fig. S5 B). To examine this phenotype without relying on RNAi knockdowns, we tagged endogenous p27dnc-6 with 3×flag and subsequently generated a genetic null allele, p27dnc-6(−) by introducing a stop/ on January 19, 2018jcb.rupress.orgDownloaded from JCB • Volume 216 • NumBer 4 • 2017950 on January 19, 2018jcb.rupress.orgDownloaded from Dynein recruitment to kinetochores • Gama et al. 951 frameshift immediately after the p27dnc-6 start codon (Fig.6A). The p27dnc-6(−) allele was maintained by introduction of a balancer chromosome containing the wild-type locus, p27dnc-6(wt). F1 p27dnc-6(−/−) progeny, which receive a maternal supply of p27DNC-6, survived to adulthood and produced F2 p27dnc-6(−/−) progeny (Fig. 6 B). This permitted phenotypic analysis in a penetrant p27dnc-6-null background. Immunoblotting of F1 p27dnc-6(−/−) adults showed that p27DNC-6 was not required for stability of the p150DNC-1 and p50DNC-2 subunits (Fig.6C), and immunofluorescence of F2 p27dnc-6(−/−) embryos revealed normal p150DNC-1 localization to the nuclear envelope, the cell cortex, and centrosomes (Fig.6D). This suggested that the dynactin complex retained its structural integrity in the absence of p27DNC-6.Live imaging of F2 p27dnc-6(−/−) embryos using differential interference contrast confirmed that p27DNC-6 is dispensable for dynactin-dependent positioning of pronuclei and centrosomes in the first mitotic division (Video3). However, despite successful bipolar spindle assembly (Fig.6E), F2 p27dnc-6(−/−) embryos became aneuploid because of chromosome missegregation (Fig.6, D and E; and Video3), providing an explanation for the penetrant lethality observed in F2 p27dnc-6(−/−) embryos. Visualization of chromosome dynamics with mCherry::histone H2B revealed severe defects in chromosome congression and segregation (Fig.6F and Video4). The defects resembled those of the SpindlySPDL-1 motif mutant F199A, which delocalizes dynein–dynactin from kinetochores (Cheerambathur et al., 2013). Because RZZ inhibits the formation of stable kinetochore–microtubule attachments when kinetochores lack dynein–dynactin, the chromosome segregation defects of SpindlySPDL-1 F199A can be ameliorated by codepleting RZZ subunits. We observed a striking rescue of chromosome congression in p27dnc-6(−/−) embryos after rod-1(RNAi), suggesting that dynein–dynactin were delocalized from kinetochores in the absence of p27DNC-6 (Fig.6F). To test this directly, we crossed the p27dnc-6(−) allele with a gfp::p50dnc-2 knock-in allele. GFP::p50DNC-2 localized to centrosomes and the mitotic spindle in F2 p27dnc-6(−/−) embryos but was not detected at kinetochores (Fig. 6 G). We then triggered maximal kinetochore accumulation of GFP::p50DNC-2 by generating monopolar spindles. GFP::p50DNC-2 prominently accumulated at microtubule-unattached kinetochores of monopolar spindles in control embryos (Fig.6H). In contrast, GFP::p50DNC-2 was absent from kinetochores in F2 p27dnc-6(−/−) embryos, despite localizing to the monopolar spindle itself. Thus, our analysis of an engineered p27dnc-6-null allele demonstrates that the dynactin pointed-end complex is essential for dynein–dynactin recruitment to kinetochores, consistent with our biochemical data and prior in vivo characterization of Spindly motif mutants in C.elegans and human cells. Spindly-like motifs implicated in dynactin pointed-end binding are present in multiple dynein adaptors Primary sequence analysis identified Spindly-like motifs in dynein adaptors involved in intracellular transport (Fig.5F). In all cases, the motif is preceded by a similarly sized coiled-coil region (260 residues on average), whose N-terminal segment also often harbors the LIC-binding CC1 box (Fig. S4 A). We found that Strep-tagged BICD22–422, which contains a Spindly-like motif at residues 336–340, pulled down the purified pointed-end complex in vitro (Fig. S4, B, C, and F). Mutating E339 to alanine (EA) weakened BICD22–422 binding to the pointed-end complex, and introducing four additional alanine mutations (5A) further diminished the interaction (Fig. S4, C and F). In contrast, BICD22–422 binding to LIC1 was not affected by the 5A mutant (Fig. S4 D). We conclude that binding of Spindly and BICD2 to dynactin’s pointed-end complex requires a motif shared by several functionally distinct dynein adaptors. Discussion The molecular motor dynein is used in virtually all cellular processes that require microtubule minus end–directed motility. Dynein’s functional diversity requires that the motor associate with cofactors and cargo-specific adaptors, but how dynein is recruited and locally activated at subcellular structures remains poorly understood. In this paper, we provide a detailed view of how dynein is targeted to the mitotic kinetochore (Fig.7A). Our results suggest that the mechanism used by the kinetochore to engage dynein is also relevant for how dynein interacts with cargo in the context of intracellular transport (Fig.7B). Our analysis of C.elegans and human RZZ identifies a conserved module, consisting of Rod’s N-terminal β-propeller in complex with the Zwilch subunit, as the kinetochore receptor of the dynein adaptor Spindly. We also show that the Rod β-propeller–Zwilch module is dispensable for RZZ targeting to kinetochores and that this function instead resides in Rod’s α-solenoid, whose Sec39 domain binds the Zw10 subunit. Zw10 is known to interact with the outer kinetochore component Zwint (Starr et al., 2000; Vos et al., 2011), which in turn associates with the C-terminal domain of Knl1 (Petrovic et al., 2010). However, Zw10 mutants that cannot bind Zwint still localize to kinetochores (Famulski et al., 2008), and depletion of Figure 4. Human Rod lacking its β-propeller localizes to kinetochores with Zw10 but fails to recruit Zwilch or Spindly. (A and B) HeLa cells coimmunostained with ACAs and Spindly/Mad1 (A) or Zw10 (B) after transfection with an siRNA oligonucleotide against hRod or luciferase (Luc) as a control. Cells were incubated in 1µM nocodazole for 4h before fixation. (C) Quantification of kinetochore levels of Spindly, Mad1, and Zw10 using immunofluorescence intensity measurements normalized to ACA signal. Each condition represents ≥50 kinetochore measurements from 10 or more different cells. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***, P < 0.0001). (D) Immunoblot of HeLa Flp-In T-REx cells with an antibody against human Rod (hRod), showing expression levels of endogenous hRod and RNAi-resistant full-length GFP::hRod (FL) or GFP::hRod lacking the β-propeller domain (Δ1–375). Luciferase siRNA was used as a control in RNAi experiments, and α-tubulin served as the loading control. Note that GFP::hRod(Δ1–375) migrates at the same size as endogenous hRod. (E) HeLa Flp-In T-REx cells expressing GFP::hRod or GFP::hRod(Δ1–375), depleted of endogenous hRod and immunostained with anti-GFP and ACAs. Blow-ups show examples of individual sister kinetochore pairs. (F) Quantification of full-length and Δ1–375 GFP::hRod levels at kinetochores as in C.(G–J) HeLa Flp-In T-REx cells immunostained for GFP and Zw10 (G), Zwilch (H), Spindly (I), or Mad1 (J) in cells expressing full-length and Δ1–375 GFP::hRod after depletion of endogenous hRod. (K) Quantification of kinetochore levels for the components in G–J as described in C using immunofluorescence intensity measurements normalized to GFP::hRod signal. (L–O) HeLa Flp-In T-REx cells in prometaphase immunostained as in G–J but without nocodazole treatment. Arrowheads point to the accumulation of GFP::hRod at spindle poles along with Zw10, Zwilch, Spindly, and Mad1. Polar accumulation is missing in cells expressing GFP::hRod(Δ1–375). Bars: (A, B, E [main images], G–J, and L–O) 5 µm; (E, zoom) 1 µm. on January 19, 2018jcb.rupress.orgDownloaded from JCB • Volume 216 • NumBer 4 • 2017958 For GST::LIC1::6×His, bacterial pellets were resuspended in lysis buffer E (50mM Hepes, 250mM NaCl, 0.1% Tween 20, 10mM EDTA, 10 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 mM benzamidine-HCl, and 1 mg/ml lysozyme, pH 8.0). GST::LIC1::6×His was purified by tandem affinity chromatography using Glutathione Agarose beads followed by HIS-Select Nickel Affinity Gel beads. Glutathione Agarose beads were incubated in batch with the cleared lysates, then were washed with wash buffer E (25mM Hepes, 250mM NaCl, 0.1% Tween 20, 1mM DTT, and 2mM benzamidine-HCl, pH 8.0), and proteins were eluted on a gravity column with elution buffer E (50mM Hepes, 150mM NaCl, 10mM reduced l-glutathione, 1mM DTT, and 2mM benzamidine-HCl, pH 8.0). Fractions containing the recombinant proteins were pooled, incubated in batch with Nickel Affinity Gel beads, and washed with wash buffer C (25mM Hepes, 250mM NaCl, 20mM imidazole, 0.1% Tween 20, 1mM DTT, and 2mM benzamidine-HCl, pH 8.0). Proteins were eluted on a gravity column with elution buffer C (50mM Hepes, 150mM NaCl, 250mM imidazole, 1mM DTT, and 2mM benzamidine-HCl, pH 8.0). Fractions containing the recombinant proteins were pooled and dialyzed against storage buffer (25mM Hepes and 150mM NaCl, pH 7.5), and glycerol and DTT were added to final concentrations of 10% (vol/vol) and 1mM, respectively. Aliquots were flash frozen in liquid nitrogen and stored at −80°C. Pull-downs from insect cell lysates. Insect cell lysates containing RZZ subunits (Fig.3, A and E) were prepared from 50ml of a 0.8 × 106 cell/ml suspension. Cells were harvested by centrifugation at 800g for 5 min. Pellets were resuspended in 2ml of pull-down buffer (50mM Hepes, 50 mM NaCl, and 5 mM DTT, pH 7.5) supplemented with EDTA-free cOmplete Protease Inhibitor Cocktail, disrupted by sonication, and cleared by centrifugation at 20,000g for 30 min. Cleared lysates were aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C. To decrease interference of endogenous glutathione-binding proteins present in insect cells, thawed lysate aliquots were first predepleted with Glutathione Agarose beads. Then, 15µl of Glutathione Agarose beads coated with 225 pmol of the GST::SpindlySPDL-1 constructs or GST only were incubated with 250µl of insect cell lysate for 1h at 4°C.Beads were washed with 3 × 500µl of pull-down buffer, and proteins were eluted in pull-down buffer supplemented with 15mM reduced l-glutathione for 15 min at room temperature. Pull-downs with purified ROD-11–372–ZwilchZWL-1 complex or ROD-11–372. 50 pmol purified ROD-11–372–ZwilchZWL-1 complex or ROD-11–372 was incubated with 50 pmol GST::SpindlySPDL-1 for 1h at 4°C in 150µl pull-down buffer (50mM Hepes, 50mM NaCl, and 5mM DTT, pH 7.5) supplemented with 15µl of Glutathione Agarose beads. Beads were washed with 3 × 500µl pull-down buffer, and proteins were eluted with pull-down buffer containing 15mM reduced l-glutathione. Pull-downs with purified LIC1. 50 pmol of GST::LIC1::6×His were incubated with 50 pmol BICD2::Strep-tag II or 250 pmol Spindly::Strep-tag II for 1 h at 4°C in 150 µl pull-down buffer (50 mM Hepes, 50mM NaCl, and 5mM DTT, pH 7.5) supplemented with 15µl of Glutathione Agarose beads. Beads were washed with 3 × 500µl pull-down buffer, and proteins were eluted with pull-down buffer containing 15mM reduced l-glutathione. For the competition experiment in Fig. S4 E, 50 pmol GST::LIC1::6×His was mixed with 250 pmol Spindly::Strep-tag II and 0, 50, 100, or 200 pmol BICD2::Strep-tag II. Pull-downs with purified dynactin pointed-end complex. 50 pmol Spindly::Strep-tag II or BICD2::Strep-tag II was incubated with 250 pmol dynactin pointed-end complex for 1h at 4°C in 150µl pulldown buffer (50mM Hepes, 100mM NaCl, 5mM DTT, and 0.1% Tween 20, pH 7.5) supplemented with 15µl of Strep-Tactin Sepharose beads. Beads were washed with 3 × 500µl pull-down buffer, and proteins were eluted with buffer E (100mM Tris/HCl, 150mM NaCl, 1mM EDTA, and 2.5mM desthiobiotin, pH 8.0; IBA). Pull-downs from porcine brain lysate. Porcine brain lysate was prepared as described previously (McKenney et al., 2014). In brief, fresh brains were broken into small chunks, flash-frozen in liquid nitrogen, and stored at −80°C.Frozen brain chunks were homogenized in equal weight/volume of buffer (50mM Hepes, 50mM Pipes, 1mM EDTA, and 2mM MgSO4, pH 7.0) using a waring blender, followed by glass pestle grinding. After clarification at 34,000 g for 45 min, the crude homogenate was flash frozen in 1-ml aliquots and stored at −80°C.For the pull-downs, 100µl of brain lysate was diluted to 500µl final volume with buffer A (30mM Hepes, 50mM K-acetate, 2mM Mg-acetate, 1mM EGTA, 10% glycerol, 1mM PMSF, 5mM DTT, and 0.1% NP-40, pH 7.4), and 50 pmol Spindly::Strep-tag II or BICD2::Strep-tag II were added together with 15 µl of Strep-Tactin Sepharose beads. After incubation for 1h at 4°C, beads were washed with 3 × 500µl buffer A, and proteins were eluted with elution buffer E (100mM Tris/HCl, 150mM NaCl, 1mM EDTA, and 2.5mM desthiobiotin, pH 8.0; IBA). Online supplemental material Fig. S1 summarizes two-hybrid experiments between RZZ subunits and describes RZZ subunit transgenes used for C.elegans strain generation. Fig. S2 extends the results in Fig.2 with additional characterization of the ZwilchZWL-1 mutant E433A/E437A, including size-exclusion chromatography experiments with purified recombinant ZwilchZWL-1 bound to the ROD-1 β-propeller and phenotypic analysis of the mutant in the early embryo. Fig. S3 shows Coomassie-stained gels corresponding to the pull-downs presented in Fig.3 (C and D). Fig. S4 shows the similarities in domain architecture between Spindly, BICD2, and additional dynein adaptors that contain a CC1 box and/or a Spindly-like motif and demonstrates that BICD2 interacts with LIC1 and dynactin’s pointed-end complex in vitro. Fig. S5 relates to Fig.6 and describes the consequences of depleting dynactin pointed-end complex subunits in the C.elegans one-cell embryo by RNAi. Video1 shows how the ZwilchZWL-1 E433A/E437A mutant displaces SpindlySPDL-1 from kinetochores. Video2 shows how the ZwilchZWL-1 E433A/E437A mutant causes chromosome segregation defects that resemble those of SpindlySPDL-1 depletions. Video3 shows the first embryonic divisions in the absence of p27DNC-6 imaged using differential interference contrast, showing normal pronuclear migration and spindle positioning but defective chromosome segregation leading to multinucleate cells. Video4 show the first embryonic division in the absence of p27DNC-6 with mCherry-labeled histone H2B to visualize chromosome segregation. Table S1 is a list of worm strains used in this study. Table S2 is a list of oligonucleotides used in this study for dsRNA production. Tables S1 and S2 are included as Excel files. Acknowledgments We wish to thank Andrea Musacchio for antibodies, Kevin Corbett for the 6×His::MBP expression vector, and Arshad Desai for generous support during the early stages of this project. This work was supported by a European Research Council Starting Grant (Dyneinome 338410) and a European Molecular Biology Organization Installation Grant to R.Gassmann. This work was also supported by funding from the Fundação para a Ciência e a Tecnologia to R. Gassmann (IF/01015/2013/CP1157/CT0006), C. Pereira (SFRH_BPD_95648_2013), and D.J. Barbosa (SFRH_ BPD_101898_2014). Some C.elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). on January 19, 2018jcb.rupress.orgDownloaded from Dynein recruitment to kinetochores • Gama et al. 959 The authors declare no competing financial interests. Author contributions: R. Gassmann, J.B. Gama, C. Pereira, P.A. Simões, and A.X. Carvalho conceived and designed experiments. J.B.Gama performed the protein biochemistry. C.Pereira and P.A.Simões performed the C.elegans work. R.Celestino constructed the dnc-6::3×flag and dnc-6–null alleles and contributed to protein purification with C.Carvalho. R.M.Reis performed the tissue culture work. D.J.Barbosa generated the GFP::DNC-2 strain and the anti– DNC-2 antibody, H.R. Pires generated the anti–DNC-1 antibody, J. Amorim performed the yeast two-hybrid experiments, and D.K.Cheerambathur generated the mCherry::ROD-1 strain and the anti–ROD-1 antibody. R. Gassmann, J.B. Gama, C. Pereira, and P.A.Simões prepared the figures and wrote the paper with advice from D.K.Cheerambathur and A.X.Carvalho. Submitted: 30 October 2016 Revised: 3 January 2017 Accepted: 23 January 2017 References Arribere, J.A., R.T.Bell, B.X.H.Fu, K.L.Artiles, P.S.Hartman, and A.Z.Fire. 2014. Efficient marker-free recovery of custom genetic modifications with CRI SPR/Cas9 in Caenorhabditis elegans. 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