Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.

In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early C. elegans embryo, cyclin B3 is the dominant driver of rapid embryonic mitoses. Metazoans typically have three cyclin B isoforms that associate with and activate Cdk1 kinase to orchestrate mitotic events: the related cyclins B1 and B2 and the more divergent cyclin B3. We show that whereas embryos expressing cyclins B1 and B2 support slow mitosis (NEBD to Anaphase ~ 600s), the presence of cyclin B3 dominantly drives the ~3-fold faster mitosis observed in wildtype embryos. CYB-1/2-driven mitosis is longer than CYB-3-driven mitosis primarily because the progression of mitotic events itself is slower, rather than delayed anaphase onset due to activation of the spindle checkpoint or inhibitory phosphorylation of the anaphase activator CDC-20. Addition of cyclin B1 to cyclin B3-only mitosis introduces an ~60s delay between the completion of chromosome alignment and anaphase onset, which likely ensures segregation fidelity; this delay is mediated by inhibitory phosphorylation on CDC-20. Thus, the dominance of cyclin B3 in driving mitotic events, coupled to introduction of a short cyclin B1-dependent delay in anaphase onset, sets the rapid pace and ensures fidelity of mitoses in the early C. elegans embryo.

BUB-1-bound PLK-1 directs CDC-20 kinetochore recruitment to ensure timely embryonic mitoses.

During mitosis, chromosomes assemble kinetochores to dynamically couple with spindle microtubules.1,2 Kinetochores also function as signaling hubs directing mitotic progression by recruiting and controlling the fate of the anaphase promoting complex/cyclosome (APC/C) activator CDC-20.3,4,5 Kinetochores either incorporate CDC-20 into checkpoint complexes that inhibit the APC/C or dephosphorylate CDC-20, which allows it to interact with and activate the APC/C.4,6 The importance of these two CDC-20 fates likely depends on the biological context. In human somatic cells, the major mechanism controlling mitotic progression is the spindle checkpoint. By contrast, progression through mitosis during the cell cycles of early embryos is largely checkpoint independent.7,8,9,10 Here, we first show that CDC-20 phosphoregulation controls mitotic duration in the C. elegans embryo and defines a checkpoint-independent temporal mitotic optimum for robust embryogenesis. CDC-20 phosphoregulation occurs at kinetochores and in the cytosol. At kinetochores, the flux of CDC-20 for local dephosphorylation requires an ABBA motif on BUB-1 that directly interfaces with the structured WD40 domain of CDC-20.6,11,12,13 We next show that a conserved "STP" motif in BUB-1 that docks the mitotic kinase PLK-114 is necessary for CDC-20 kinetochore recruitment and timely mitotic progression. The kinase activity of PLK-1 is required for CDC-20 to localize to kinetochores and phosphorylates the CDC-20-binding ABBA motif of BUB-1 to promote BUB-1-CDC-20 interaction and mitotic progression. Thus, the BUB-1-bound pool of PLK-1 ensures timely mitosis during embryonic cell cycles by promoting CDC-20 recruitment to the vicinity of kinetochore-localized phosphatase activity.

Nuclear-enriched protein phosphatase 4 ensures outer kinetochore assembly prior to nuclear dissolution.

A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Important mitotic events occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here, we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant bi-orientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4-CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.

TMEM161B modulates radial glial scaffolding in neocortical development.

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria (PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Spindle assembly checkpoint activation and silencing at kinetochores.

The spindle assembly checkpoint (SAC) is a surveillance mechanism that promotes accurate chromosome segregation in mitosis. The checkpoint senses the attachment state of kinetochores, the proteinaceous structures that assemble onto chromosomes in mitosis in order to mediate their interaction with spindle microtubules. When unattached, kinetochores generate a diffusible inhibitor that blocks the activity of the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase required for sister chromatid separation and exit from mitosis. Work from the past decade has greatly illuminated our understanding of the mechanisms by which the diffusible inhibitor is assembled and how it inhibits the APC/C. However, less is understood about how SAC proteins are recruited to kinetochores in the absence of microtubule attachment, how the kinetochore catalyzes formation of the diffusible inhibitor, and how attachments silence the SAC at the kinetochore. Here, we summarize current understanding of the mechanisms that activate and silence the SAC at kinetochores and highlight open questions for future investigation.

The N-terminal tail of C. elegans CENP-A interacts with KNL-2 and is essential for centromeric chromatin assembly.

Centromeres are epigenetically defined by the centromere-specific histone H3 variant CENP-A. Specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, with CENP-A-dependent centromeres. Here, we show that the extended N-terminal tail of Caenorhabditis elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly; removal of this region prevents CENP-A loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-tail containing the predicted structured region binds to KNL-2, a conserved SANTA domain and Myb domain-containing protein (referred to as M18BP1 in vertebrates) specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode Caenorhabditis briggsae, despite divergence of the N-tail and KNL-2 primary sequences. Thus, the extended N-tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates a direct interaction between CENP-A and KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.

The PHLPP1 N-Terminal Extension Is a Mitotic Cdk1 Substrate and Controls an Interactome Switch.

PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the prosurvival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here, we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis, PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE and gains proximity to kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor-suppressive function.

A tripartite mechanism catalyzes Mad2-Cdc20 assembly at unattached kinetochores.

During cell division, kinetochores couple chromosomes to spindle microtubules. To protect against chromosome gain or loss, kinetochores lacking microtubule attachment locally catalyze association of the checkpoint proteins Cdc20 and Mad2, which is the key event in the formation of a diffusible checkpoint complex that prevents mitotic exit. We elucidated the mechanism of kinetochore-catalyzed Mad2-Cdc20 assembly with a probe that specifically monitors this assembly reaction at kinetochores in living cells. We found that catalysis occurs through a tripartite mechanism that includes localized delivery of Mad2 and Cdc20 substrates and two phosphorylation-dependent interactions that geometrically constrain their positions and prime Cdc20 for interaction with Mad2. These results reveal how unattached kinetochores create a signal that ensures genome integrity during cell division.

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes.

Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.

A Non-canonical BRCT-Phosphopeptide Recognition Mechanism Underlies RhoA Activation in Cytokinesis.

Cytokinesis partitions the cell contents to complete mitosis. During cytokinesis, polo-like kinase 1 (PLK1) activates the small GTPase RhoA to assemble a contractile actomyosin ring. PLK1 is proposed to pattern RhoA activation by creating a docking site on the central spindle that concentrates the RhoA guanine nucleotide exchange factor ECT2. However, ECT2 targeting to the central spindle is dispensable for cytokinesis, indicating that how PLK1 controls RhoA activation remains unresolved. To address this question, we employed an unbiased approach targeting ∼100 predicted PLK1 sites in two RhoA regulators: ECT2 and the centralspindlin complex, composed of CYK4 and kinesin-6. This comprehensive approach suggested that the only functionally critical PLK1 target sites are in a single cluster in the CYK4 N terminus. Phosphorylation of this cluster promoted direct interaction of CYK4 with the BRCT repeat module of ECT2. However, mutational analysis in vitro and in vivo led to the surprising finding that the interaction was independent of the conserved "canonical" residues in ECT2's BRCT repeat module that, based on structurally characterized BRCT-phosphopeptide interactions, were presumed critical for binding. Instead, we show that the ECT2 BRCT module binds phosphorylated CYK4 via a distinct conserved basic surface. Basic surface mutations mimic the effects on cytokinesis of loss of CYK4 cluster phosphorylation or inhibition of PLK1 activity. Together with evidence for ECT2 autoinhibition limiting interaction with CYK4 in the cytoplasm, these results suggest that a spatial gradient of phosphorylated CYK4 around the central spindle patterns RhoA activation by interacting with ECT2 on the adjacent plasma membrane.

Rashomon at the kinetochore: Function(s) of the Mad1-cyclin B1 complex.

In the film Rashomon, four witnesses describe seemingly contradictory views of one event. In a recent analogy, an interaction between the master mitotic regulator cyclin B1 and the spindle checkpoint component Mad1 was independently described by three groups who propose strikingly different functions for this interaction. Here, we summarize their findings and present a perspective on reconciling the different views.

The G2-to-M Transition Is Ensured by a Dual Mechanism that Protects Cyclin B from Degradation by Cdc20-Activated APC/C.

In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. Here, we show that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition.

Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan.

Phenotypic screening has identified small-molecule modulators of aging, but the mechanism of compound action often remains opaque due to the complexities of mapping protein targets in whole organisms. Here, we combine a library of covalent inhibitors with activity-based protein profiling to coordinately discover bioactive compounds and protein targets that extend lifespan in Caenorhabditis elegans. We identify JZL184-an inhibitor of the mammalian endocannabinoid (eCB) hydrolase monoacylglycerol lipase (MAGL or MGLL)-as a potent inducer of longevity, a result that was initially perplexing as C. elegans does not possess an MAGL ortholog. We instead identify FAAH-4 as a principal target of JZL184 and show that this enzyme, despite lacking homology with MAGL, performs the equivalent metabolic function of degrading eCB-related monoacylglycerides in C. elegans. Small-molecule phenotypic screening thus illuminates pure pharmacological connections marking convergent metabolic functions in distantly related organisms, implicating the FAAH-4/monoacylglyceride pathway as a regulator of lifespan in C. elegans.

Employing the one-cell C. elegans embryo to study cell division processes.

The one-cell Caenorhabditis elegans embryo offers many advantages for mechanistic analysis of cell division processes. Conservation of key genes and pathways involved in cell division makes findings in C. elegans broadly relevant. A key technical advantage of this system is the ability to penetrantly deplete essential gene products by RNA interference (RNAi) and replace them with wild-type or mutant versions expressed at endogenous levels from single copy RNAi-resistant transgene insertions. This ability to precisely perturb essential genes is complemented by the inherently highly reproducible nature of the zygotic division that facilitates development of quantitative imaging assays. Here, we detail approaches to generate targeted single copy transgene insertions that are RNAi-resistant, to engineer variants of individual genes employing transgene insertions as well as at the endogenous locus, and to in situ tag genes with fluorophores/purification tags. We also describe imaging assays and common image analysis tools employed to quantitatively monitor phenotypic effects of specific perturbations on meiotic and mitotic chromosome segregation, centrosome assembly/function, and cortical dynamics/cytokinesis.

Taming the Beast: Control of APC/CCdc20-Dependent Destruction.

The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit ubiquitin ligase that triggers the metaphase-to-anaphase transition in the cell cycle by targeting the substrates cyclin B and securin for destruction. APC/C activity toward these two key substrates requires the coactivator Cdc20. To ensure that cells enter mitosis and partition their duplicated genome with high accuracy, APC/CCdc20 activity must be tightly controlled. Here, we discuss the mechanisms that regulate APC/CCdc20 activity both before and during mitosis. We focus our discussion primarily on the chromosomal pathways that both accelerate and delay APC/C activation by targeting Cdc20 to opposing fates. The findings discussed provide an overview of how cells control the activation of this major cell cycle regulator to ensure both accurate and timely cell division.

Kinetochores accelerate or delay APC/C activation by directing Cdc20 to opposing fates.

Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.