The spindle assembly checkpoint (SAC) delays mitotic progression when chromosomes are not properly attached to microtubules of the mitotic spindle. Cells vary widely in the extent to which they delay ...mitotic progression upon SAC activation. To explore the mechanisms that determine checkpoint strength in different cells, we systematically measured the mitotic delay induced by microtubule disruption at different stages of embryogenesis in Caenorhabditis elegans. Strikingly, we observed a gradual increase in SAC strength after each round of division. Analysis of mutants that alter cell size or ploidy revealed that SAC strength is determined primarily by cell size and the number of kinetochores. These findings provide clear evidence in vivo that the kinetochore-to-cytoplasm ratio determines the strength of the SAC, providing new insights into why cells exhibit such large variations in their SAC responses.
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•In C. elegans embryos, small cells have a stronger spindle assembly checkpoint•Checkpoint strength increases in mutants with small cell size or increased ploidy•The amount of checkpoint-generating kinetochore signal is unaffected by cell size•Checkpoint strength is determined by the ratio of kinetochore signal to cytoplasm
The spindle assembly checkpoint (SAC) delays mitotic progression when there are unattached kinetochores, but strength of the block varies between cell types. Galli and Morgan show that the kinetochore-to-cytoplasm ratio determines checkpoint strength. During C. elegans embryogenesis, SAC strength increases after each round of division due to decreasing cell size.
The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase that polyubiquitinates specific substrates at precise times in the cell cycle, thereby triggering the events of late mitosis ...in a strict order. The robust substrate specificity of the APC/C prevents the potentially deleterious degradation of non-APC/C substrates and also averts the cell-cycle errors and genomic instability that could result from mistimed degradation of APC/C targets. The APC/C recognizes short linear sequence motifs, or degrons, on its substrates. The specific and timely modification and degradation of APC/C substrates is likely to be modulated by variations in degron sequence and context. We discuss the extensive affinity, specificity, and selectivity determinants encoded in APC/C degrons, and we describe some of the extrinsic mechanisms that control APC/C-substrate recognition. As an archetype for protein motif-driven regulation of cell function, the APC/C-substrate interaction provides insights into the general properties of post-translational regulatory systems.
Davey and Morgan review our current knowledge of the short linear sequence motifs, or degrons, that mediate specific and selective substrate binding to the ubiquitin ligase APC/C, and they discuss how variations in degron features and extrinsic regulation determine the timing of APC/C substrate degradation in mitosis.
The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein ...mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein’s transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.
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•Nucleocapsid protein of SARS-CoV-2 forms biomolecular condensates with viral RNA•Unmodified N protein forms gel-like condensates containing discrete RNP particles•Phosphorylated N protein forms dynamic, liquid-like condensates•The two condensate forms are well suited for the two major functions of N protein
Carlson et al. demonstrate that the nucleocapsid (N) protein of SARS-CoV-2, together with viral RNA, forms gel-like biomolecular condensates and particles that are consistent with its genome-packaging role. Phosphorylation transforms condensates into liquid-like droplets, which may provide a cytoplasmic compartment to support the protein’s function in viral genome transcription.
The fidelity of chromosome segregation depends on the spindle assembly checkpoint (SAC). In the presence of unattached kinetochores, anaphase is delayed when three SAC components (Mad2, Mad3/BubR1, ...and Bub3) inhibit Cdc20, the activating subunit of the anaphase-promoting complex (APC/C). We analyzed the role of Cdc20 autoubiquitination in the SAC of budding yeast. Reconstitution with purified components revealed that a Mad3-Bub3 complex synergizes with Mad2 to lock Cdc20 on the APC/C and stimulate Cdc20 autoubiquitination, while inhibiting ubiquitination of substrates. SAC-dependent Cdc20 autoubiquitination required the Mnd2/Apc15 subunit of the APC/C. General inhibition of Cdc20 ubiquitination in vivo resulted in high Cdc20 levels and a failure to establish a SAC arrest, suggesting that SAC establishment depends on low Cdc20 levels. Specific inhibition of SAC-dependent ubiquitination, by deletion of Mnd2, allowed establishment of a SAC arrest but delayed release from the arrest, suggesting that Cdc20 ubiquitination is also required for SAC inactivation.
► Spindle checkpoint proteins synergize to stimulate Cdc20 autoubiquitination ► Mnd2/Apc15 is required for Cdc20 autoubiquitination in the checkpoint ► Cdc20 stabilization throughout the cycle prevents spindle checkpoint establishment ► Cells lacking Mnd2 maintain a checkpoint arrest but delay checkpoint inactivation
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of ...these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
► Cyclins gradually raise the active-site specificity of Cdk1 during the cell cycle ► A novel substrate docking motif is specific for G1-cyclin-Cdk1 complexes ► G1 cyclin-Cdk1 shows distinct features of substrate site consensus specificity ► Several G1, G2, and M phase-specific Cdk1 targets exist in the cell
Transient interactions between the anaphase-promoting complex/cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the ...order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates.
In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex
. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase ...that cleaves the cohesin subunit SCC1 (also known as RAD21
). Separase is activated by degradation of its inhibitors, securin
and cyclin B
, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans
and yeast
, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK1
. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine
that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding ...proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.
Accurate genome segregation depends on cohesion mechanisms that link duplicated sister chromatids, thereby allowing their tension-dependent biorientation in metaphase. In
Saccharomyces cerevisiae, ...cohesion is established during DNA replication when Eco1 acetylates the cohesin subunit Smc3. Cohesion establishment is restricted to S phase of the cell cycle, but the molecular basis of this regulation is unknown. Here, we show that Eco1 is negatively regulated by the protein kinase Cdk1. Phosphorylation of Eco1 after S phase targets it to SCF
Cdc4 for ubiquitination and subsequent degradation. A nonphosphorylatable mutant of Eco1 establishes cohesion after DNA replication, suggesting that Cdk1-dependent phosphorylation of Eco1 is a key factor limiting establishment to S phase. We also show that deregulation of Eco1 results in chromosome separation defects in anaphase. We conclude that this regulatory mechanism helps optimize the level of sister chromatid cohesion, ensuring a robust and efficient anaphase.
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► Eco1 phosphorylation by Cdk1 in late S phase targets it for degradation via SCF
Cdc4 ► Eco1 that cannot be phosphorylated can establish cohesion postreplication ► Unregulated Eco1 activity affects timing of sister chromatid separation
Robust progression through the cell-division cycle depends on the precisely ordered phosphorylation of hundreds of different proteins by cyclin-dependent kinases (CDKs) and other kinases. The order ...of CDK substrate phosphorylation depends on rising CDK activity, coupled with variations in substrate affinities for different CDK-cyclin complexes and the opposing phosphatases 1–4. Here, we address the ordering of substrate phosphorylation by a second major cell-cycle kinase, Cdc7-Dbf4 or Dbf4-dependent kinase (DDK). The primary function of DDK is to initiate DNA replication by phosphorylating the Mcm2-7 replicative helicase 5–7. DDK also phosphorylates the cohesin acetyltransferase Eco1 8. Sequential phosphorylations of Eco1 by CDK, DDK, and Mck1 create a phosphodegron that is recognized by the ubiquitin ligase SCFCdc4. DDK, despite being activated in early S phase, does not phosphorylate Eco1 to trigger its degradation until late S phase 8. DDK associates with docking sites on loaded Mcm double hexamers at unfired replication origins 9, 10. We hypothesized that these docking interactions sequester limiting amounts of DDK, delaying Eco1 phosphorylation by DDK until replication is complete. Consistent with this hypothesis, we find that overproduction of DDK leads to premature Eco1 degradation. Eco1 degradation also occurs prematurely if Mcm complex loading at origins is prevented by depletion of Cdc6, and Eco1 is stabilized if loaded Mcm complexes are prevented from firing by a Cdc45 mutant. We propose that the timing of Eco1 phosphorylation, and potentially that of other DDK substrates, is determined in part by sequestration of DDK at unfired replication origins during S phase.
•Dbf4-Cdc7 (DDK) phosphorylates Eco1 in late S phase, triggering Eco1 degradation•DDK activity toward Eco1 is limiting during S phase•DDK is sequestered during S phase by Mcm complexes at unfired replication origins
Seoane and Morgan explore the mechanism underlying the cell-cycle timing of Eco1 phosphorylation, and they present evidence that the protein kinase Dbf4-Cdc7 does not phosphorylate Eco1 until late S phase because the kinase is sequestered during S phase by Mcm complexes at unfired replication origins.
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