A new histone at the centromere?

The centromere is a classic system to study epigenetic specification, and most research has focused on a specialized histone variant, CENP-A, that is required for kinetochore assembly. Now Nishino et al. reveal a new level of complexity for centromeric chromatin, by showing that the kinetochore complex CENP-T-W-S-X shares structural and functional properties with canonical histones.

Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro.

The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.

Protein architecture of the human kinetochore microtubule attachment site.

Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

BACKGROUND: Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. METHODS: We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). RESULTS: We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. CONCLUSIONS: Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

A Centromere-Signaling Network Underlies the Coordination among Mitotic Events.

There is increasing evidence that regulators of the spindle checkpoint, kinetochore-microtubule attachments, and sister chromatid cohesion are part of an interconnected mitotic regulatory circuit with two positive feedback loops and the chromosome passenger complex (CPC) at its center. If true, this conceptual breakthrough needs to be integrated into models of mitosis. In this review, we describe this circuit and point out how the double feedback loops could provide insights into the self-organization of some mitotic processes and the autonomy of every chromosome on the mitotic spindle. We also provide working models for how mitotic events may be coordinated by this circuit.

A conserved role for COMA/CENP-H/I/N kinetochore proteins in the spindle checkpoint.

The COMA/CENP-H/I kinetochore complex regulates microtubule dynamics at kinetochores. The complex is also required to generate spindle checkpoint signals in both yeast and human cells under conditions where Aurora B activity is compromised. Our data explain why mammalian cells treated with Aurora inhibitors still have a functional spindle assembly checkpoint (SAC), since the checkpoint signals through CENP-H/I/N. The SAC effect from depleting the CENP-H/I/N complex cannot be explained by a weakened SAC signal, and the complex has no role in the SAC response to paclitaxel. We propose a model to explain the differential response of human cells to nocodazole and paclitaxel.

P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles.

Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.

A complex of p190RhoGAP-A and anillin modulates RhoA-GTP and the cytokinetic furrow in human cells.

The cytokinetic furrow is organized by the RhoA GTPase, which recruits actin and myosin II to the furrow and drives contractility. Here, we show that the RhoA GTPase-activting protein (GAP) p190RhoGAP-A (also known as ARHGAP35) has a role in cytokinesis and is involved in regulating levels of RhoA-GTP and contractility. Cells depleted of p190RhoGAP-A accumulate high levels of RhoA-GTP and markers of high RhoA activity in the furrow, resulting in failure of the cytokinetic furrow to progress to abscission. The loss of p190RhoGAP-A can be rescued by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis because they have too much myosin activity. p190RhoGAP-A binds the cytokinetic organizer anillin, and mutants of p190RhoGAP-A that are unable to bind anillin or unable to inactivate RhoA fail to rescue cytokinesis defects in p190RhoGAP-A-depleted cells. Taken together, these data demonstrate that a complex of p190RhoGAP-A and anillin modulates RhoA-GTP levels in the cytokinetic furrow to ensure progression of cytokinesis.

The Xenopus ORFeome: A resource that enables functional genomics.

Functional characterisation of proteins and large-scale, systems-level studies are enabled by extensive sets of cloned open reading frames (ORFs) in an easily-accessible format that enables many different applications. Here we report the release of the first stage of the Xenopus ORFeome, which contains 8673 ORFs from the Xenopus Gene Collection (XGC) for Xenopus laevis, cloned into a Gateway® donor vector enabling rapid in-frame transfer of the ORFs to expression vectors. This resource represents an estimated 7871 unique genes, approximately 40% of the non-redundant X. laevis gene complement, and includes 2724 genes where the human ortholog has an association with disease. Transfer into the Gateway system was validated by 5' and 3' end sequencing of the entire collection and protein expression of a set of test clones. In a parallel process, the underlying ORF predictions from the original XGC collection were re-analysed to verify quality and full-length status, identifying those proteins likely to exhibit truncations when translated. These data are integrated into Xenbase, the Xenopus community database, which associates genomic, expression, function and human disease model metadata to each ORF, enabling end-users to search for ORFeome clones with links to commercial distributors of the collection. When coupled with the experimental advantages of Xenopus eggs and embryos, the ORFeome collection represents a valuable resource for functional genomics and disease modelling.

Connecting the microtubule attachment status of each kinetochore to cell cycle arrest through the spindle assembly checkpoint.

Kinetochores generate a signal that inhibits anaphase progression until every kinetochore makes proper attachments to spindle microtubules. This spindle assembly checkpoint (SAC) increases the fidelity of chromosome segregation. We will review the molecular mechanisms by which kinetochores generate the SAC and extinguish the signal after making proper attachments, with the goal of identifying unanswered questions and new research directions. We will emphasize recent breakthroughs in how phosphorylation changes drive the activation and inhibition of the signal. We will also emphasize the dramatic changes in kinetochore structure that occur after attaching to microtubules and how these coordinate SAC function with microtubule attachment status. Finally, we will review the emerging cross talk between the DNA damage response and the SAC.

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient.

Proper partitioning of the contents of a cell between two daughters requires integration of spatial and temporal cues. The anaphase array of microtubules that self-organize at the spindle midzone contributes to positioning the cell-division plane midway between the segregating chromosomes. How this signalling occurs over length scales of micrometres, from the midzone to the cell cortex, is not known. Here we examine the anaphase dynamics of protein phosphorylation by aurora B kinase, a key mitotic regulator, using fluorescence resonance energy transfer (FRET)-based sensors in living HeLa cells and immunofluorescence of native aurora B substrates. Quantitative analysis of phosphorylation dynamics, using chromosome- and centromere-targeted sensors, reveals that changes are due primarily to position along the division axis rather than time. These dynamics result in the formation of a spatial phosphorylation gradient early in anaphase that is centred at the spindle midzone. This gradient depends on aurora B targeting to a subpopulation of microtubules that activate it. Aurora kinase activity organizes the targeted microtubules to generate a structure-based feedback loop. We propose that feedback between aurora B kinase activation and midzone microtubules generates a gradient of post-translational marks that provides spatial information for events in anaphase and cytokinesis.

Structural basis for the phase separation of the chromosome passenger complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Aurora kinases: Generators of spatial control during mitosis.

Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle.

Structural Basis for the Phase Separation of the Chromosome Passenger Complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium-exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters in Xenopus extracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

Integrative single-cell meta-analysis reveals disease-relevant vascular cell states and markers in human atherosclerosis.

Coronary artery disease (CAD) is characterized by atherosclerotic plaque formation in the arterial wall. CAD progression involves complex interactions and phenotypic plasticity among vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but human cell phenotypes remain controversial. Here, we perform an integrated meta-analysis of 22 scRNA-seq libraries to generate a comprehensive map of human atherosclerosis with 118,578 cells. Besides characterizing granular cell-type diversity and communication, we leverage this atlas to provide insights into smooth muscle cell (SMC) modulation. We integrate genome-wide association study data and uncover a critical role for modulated SMC phenotypes in CAD, myocardial infarction, and coronary calcification. Finally, we identify fibromyocyte/fibrochondrogenic SMC markers (LTBP1 and CRTAC1) as proxies of atherosclerosis progression and validate these through omics and spatial imaging analyses. Altogether, we create a unified atlas of human atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

Insights into the inhibition of the p90 ribosomal S6 kinase (RSK) by the flavonol glycoside SL0101 from the 1.5 Å crystal structure of the N-terminal domain of RSK2 with bound inhibitor.

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-L-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 Å resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

Penetration of antibody-opsonized cells by the membrane attack complex of complement promotes Ca(2+) influx and induces streamers.

We have reported that during complement-mediated cytolysis of B cells promoted by the CD20 mAbs rituximab or ofatumumab (OFA), long, thin structures that we call streamers (≥ 3 cell diameters) are rapidly generated and grow out from the cell surface. Streamers appear before cells are killed and contain opsonizing mAbs and membrane lipids. By exploiting the differential Ca(2+) requirements of discrete steps in the complement cascade, we determined that mAb-opsonized cells first tagged with C3b using C5-depleted serum are killed on addition of serum and EDTA, but the cells do not produce streamers. Also, cells first opsonized with OFA are lysed in serum containing Mg-EGTA by the alternative complement pathway but streamers are not produced. These findings indicate that Ca(2+) influx is necessary for streamer formation. Other mAbs that promote complement-mediated cytolysis also induce streamers on target cells. Streamer-like structures called nanotubes have been reported in several cellular systems, and are thought to promote intercellular communication/signaling. We tested whether this signaling could influence the susceptibility of neighboring cells contacted by streamers to complement attack and found that complement-mediated cytolysis of OFA-opsonized cells increases the resistance of unopsonized indicator cell populations to subsequent lysis when these cells are exposed to OFA and complement.

The Ndc80 complex: integrating the kinetochore's many movements.

The Ndc80 complex lies at the heart of the kinetochore, a large protein machine that accurately segregates chromosomes during cell division. The Ndc80 complex has structural roles in assembling the kinetochore, but also functions to congress chromosomes and to signal the spindle checkpoint. It directly binds to microtubules and is currently the best candidate for the long-sought protein that couples microtubule depolymerization to chromosome movement. A combination of structural and genetic data has recently converged to generate the first models for this fascinating motor activity. Additionally, recent data point to an increasingly dynamic role for Ndc80 in the kinetochore-one which involves not only simple binding to microtubules but also shifts in complex shape and its location within the overall kinetochore structure. In this review, we discuss recent advances in our understanding of the Ndc80 complex and address future areas of research.

The reversibility of mitotic exit in vertebrate cells.

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.