Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor.

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

A validation strategy to assess the role of phase separation as a determinant of macromolecular localization.

Liquid-liquid phase separation (LLPS) of putative assembly scaffolds has been proposed to drive the biogenesis of membraneless compartments. LLPS scaffolds are usually identified through in vitro LLPS assays with single macromolecules (homotypic), but the predictive value of these assays remains poorly characterized. Here, we apply a strategy to evaluate the robustness of homotypic LLPS assays. When applied to the chromosomal passenger complex (CPC), which undergoes LLPS in vitro and localizes to centromeres to promote chromosome biorientation, LLPS propensity in vitro emerged as an unreliable predictor of subcellular localization. In vitro CPC LLPS in aqueous buffers was enhanced by commonly used crowding agents. Conversely, diluted cytomimetic media dissolved condensates of the CPC and of several other proteins. We also show that centromeres do not seem to nucleate LLPS, nor do they promote local, spatially restrained LLPS of the CPC. Our strategy can be adapted to purported LLPS scaffolds of other membraneless compartments.

Structure of the human KMN complex and implications for regulation of its assembly.

Biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell's chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. One of the outer layer main components is a ten-subunit assembly comprising Knl1C, Mis12C and Ndc80C (KMN) subcomplexes. The KMN is highly elongated and docks on kinetochores and microtubules through interfaces at its opposite extremes. Here, we combine cryogenic electron microscopy reconstructions and AlphaFold2 predictions to generate a model of the human KMN that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments, we demonstrate that this mutual organization strongly stabilizes the KMN assembly. Our work thus reports a comprehensive structural and functional analysis of this part of the kinetochore microtubule-binding machinery and elucidates the path of connections from the chromatin-bound components to the force-generating components.

RZZ-Spindly and CENP-E form an integrated platform to recruit dynein to the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

Stable kinetochore-microtubule attachment requires loop-dependent Ndc80-Ndc80 binding.

During cell division, kinetochores link chromosomes to spindle microtubules. The Ndc80 complex, a crucial microtubule binder, populates each kinetochore with dozens of copies. Whether adjacent Ndc80 complexes cooperate to promote microtubule binding remains unclear. Here we demonstrate that the Ndc80 loop, a short sequence that interrupts the Ndc80 coiled-coil at a conserved position, folds into a more rigid structure than previously assumed and promotes direct interactions between full-length Ndc80 complexes on microtubules. Mutations in the loop impair these Ndc80-Ndc80 interactions, prevent the formation of force-resistant kinetochore-microtubule attachments, and cause cells to arrest in mitosis for hours. This arrest is not due to an inability to recruit the kinetochore-microtubule stabilizing SKA complex and cannot be overridden by mutations in the Ndc80 tail that strengthen microtubule attachment. Thus, loop-mediated organization of adjacent Ndc80 complexes is crucial for stable end-on kinetochore-microtubule attachment and spindle assembly checkpoint satisfaction.

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex.

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2.

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis.

Strain stiffening of Ndc80 complexes attached to microtubule plus ends.

In the mitotic spindle, microtubules attach to chromosomes via kinetochores. The microtubule-binding Ndc80 complex is an integral part of kinetochores, and is essential for kinetochores to attach to microtubules and to transmit forces from dynamic microtubule ends to the chromosomes. The Ndc80 complex has a rod-like appearance with globular domains at its ends that are separated by a long coiled coil. Its mechanical properties are considered important for the dynamic interaction between kinetochores and microtubules. Here, we present a novel method that allows us to time trace the effective stiffness of Ndc80 complexes following shortening microtubule ends against applied force in optical trap experiments. Applying this method to wild-type Ndc80 and three variants (calponin homology (CH) domains mutated or Hec1 tail unphosphorylated, phosphorylated, or truncated), we reveal that each variant exhibits strain stiffening; i.e., the effective stiffness increases under tension that is built up by a depolymerizing microtubule. The strain stiffening relation is roughly linear and independent of the state of the microtubule. We introduce structure-based models that show that the strain stiffening can be traced back to the specific architecture of the Ndc80 complex with a characteristic flexible kink, to thermal fluctuations of the microtubule, and to the bending elasticity of flaring protofilaments, which exert force to move the Ndc80 complexes. Our model accounts for changes in the amount of load-bearing attachments at various force levels and reproduces the roughly linear strain stiffening behavior, highlighting the importance of force-dependent binding affinity.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport.

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3's interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3's RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bidirectional organelle transport.

Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization.

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. A 16-subunit complex, the constitutive centromere associated network (CCAN), connects between the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, and the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has implications for the mechanism of specific recognition of the CENP-A nucleosome. A model consistent with our structure depicts the CENP-C-bound nucleosome as connected to the CCAN through extended, flexible regions of CENP-C. An alternative model identifies both CENP-C and CENP-N as specificity determinants but requires CENP-N to bind CENP-A in a mode distinct from the classical nucleosome octamer.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores.

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein-dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl-binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

On the role of phase separation in the biogenesis of membraneless compartments.

Molecular mechanistic biology has ushered us into the world of life's building blocks, revealing their interactions in macromolecular complexes and inspiring strategies for detailed functional interrogations. The biogenesis of membraneless cellular compartments, functional mesoscale subcellular locales devoid of strong internal order and delimiting membranes, is among mechanistic biology's most demanding current challenges. A developing paradigm, biomolecular phase separation, emphasizes solvation of the building blocks through low-affinity, weakly adhesive unspecific interactions as the driver of biogenesis of membraneless compartments. Here, I discuss the molecular underpinnings of the phase separation paradigm and demonstrate that validating its assumptions is much more challenging than hitherto appreciated. I also discuss that highly specific interactions, rather than unspecific ones, appear to be the main driver of biogenesis of subcellular compartments, while phase separation may be harnessed locally in selected instances to generate material properties tailored for specific functions, as exemplified by nucleocytoplasmic transport.

Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes.

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).

Phosphorylation tunes elongation propensity and cohesiveness of INCENP's intrinsically disordered region.

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP's IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP's IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP's function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined "dog-leash" model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.

Targeted substrate loop insertion by VCP/p97 during PP1 complex disassembly.

The AAA-ATPase VCP/p97/Cdc48 unfolds proteins by threading them through its central pore, but how substrates are recognized and inserted into the pore in diverse pathways has remained controversial. Here, we show that p97, with its adapter p37, binds an internal recognition site (IRS) within inhibitor-3 (I3) and then threads a peptide loop into its channel to strip I3 off protein phosphatase-1 (PP1). Of note, the IRS is adjacent to the prime interaction site of I3 to PP1, and IRS mutations block I3 processing both in vitro and in cells. In contrast, amino- and carboxy-terminal regions of I3 are not required, and even circularization of I3 does not prevent I3 processing. This was confirmed by an in vitro Förster resonance energy transfer assay that allowed kinetic analysis of the reaction. Thus, our data uncover how PP1 is released from its inhibitory partner for activation and demonstrate a remarkable plasticity in substrate threading by p97.

Assembly principles and stoichiometry of a complete human kinetochore module.

Centromeres are epigenetically determined chromosomal loci that seed kinetochore assembly to promote chromosome segregation during cell division. CENP-A, a centromere-specific histone H3 variant, establishes the foundations for centromere epigenetic memory and kinetochore assembly. It recruits the constitutive centromere-associated network (CCAN), which in turn assembles the microtubule-binding interface. How the specific organization of centromeric chromatin relates to kinetochore assembly and to centromere identity through cell division remains conjectural. Here, we break new ground by reconstituting a functional full-length version of CENP-C, the largest human CCAN subunit and a blueprint of kinetochore assembly. We show that full-length CENP-C, a dimer, binds stably to two nucleosomes and permits further assembly of all other kinetochore subunits in vitro with relative ratios closely matching those of endogenous human kinetochores. Our results imply that human kinetochores emerge from clustering multiple copies of a fundamental module and may have important implications for transgenerational inheritance of centromeric chromatin.

Overlap of NatA and IAP substrates implicates N-terminal acetylation in protein stabilization.

SMAC/DIABLO and HTRA2 are mitochondrial proteins whose amino-terminal sequences, known as inhibitor of apoptosis binding motifs (IBMs), bind and activate ubiquitin ligases known as inhibitor of apoptosis proteins (IAPs), unleashing a cell's apoptotic potential. IBMs comprise a four-residue, loose consensus sequence, and binding to IAPs requires an unmodified amino terminus. Closely related, IBM-like N termini are present in approximately 5% of human proteins. We show that suppression of the N-alpha-acetyltransferase NatA turns these cryptic IBM-like sequences into very efficient IAP binders in cell lysates and in vitro and ultimately triggers cellular apoptosis. Thus, amino-terminal acetylation of IBM-like motifs in NatA substrates shields them from IAPs. This previously unrecognized relationship suggests that amino-terminal acetylation is generally protective against protein degradation in human cells. It also identifies IAPs as agents of a general quality control mechanism targeting unacetylated rogues in metazoans.