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By live imaging and laser microsurgery, Maiato and colleagues characterize how the chromokinesin, dynein and CENP-E motor proteins cooperate to congress chromosomes peripheral to the spindle poles of the metaphase plate in mitosis. Accurate chromosome segregation during cell division in metazoans relies on proper chromosome congression at the equator. Chromosome congression is achieved after bi-orientation to both spindle poles shortly after nuclear envelope breakdown, or by the coordinated action of motor proteins that slide misaligned chromosomes along pre-existing spindle microtubules 1 . These proteins include the minus-end-directed kinetochore motor dynein 2 , 3 , 4 , 5 , and the plus-end-directed motors CENP-E at kinetochores 6 , 7 and chromokinesins on chromosome arms 8 , 9 , 10 , 11 . However, how these opposite and spatially distinct activities are coordinated to drive chromosome congression remains unknown. Here we used RNAi, chemical inhibition, kinetochore tracking and laser microsurgery to uncover the functional hierarchy between kinetochore and arm-associated motors, exclusively required for congression of peripheral polar chromosomes in human cells. We show that dynein poleward force counteracts chromokinesins to prevent stabilization of immature/incorrect end-on kinetochore–microtubule attachments and random ejection of polar chromosomes. At the poles, CENP-E becomes dominant over dynein and chromokinesins to bias chromosome ejection towards the equator. Thus, dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces mediated by chromokinesins from working in the wrong direction.

Enzymes that detyrosinate the microtubule cytoskeleton are identified Microtubules are cytoskeletal filaments that drive chromosome segregation during cell division, control cell shape and motility, and serve as rails for motor protein-based intracellular transport. Microtubules are polymers built of highly conserved subunits, α- and β-tubulin, which contain a globular core and more variable C-terminal tails that are exposed at the microtubule surface. Although microtubules are structurally uniform, they display functional specialization due to the combination of different tubulin isoforms and multiple posttranslational modifications ( 1 ). Many of these modifications occur within the C-terminal tails and affect microtubule interactions with motor proteins or regulatory factors. The first tubulin modifications were discovered more than 40 years ago and consist of the catalytic removal and reincorporation of the C-terminal tyrosine, an amino acid residue that is present in most α-tubulin isotypes (see the figure) ( 2 – 4 ). Whereas retyrosination of soluble tubulin is known to be mediated by tubulin-tyrosine ligase ( 5 ), α-tubulin detyrosination, which occurs preferentially on microtubules, is mediated by an unknown carboxypeptidase activity. On pages 1448 and 1453 of this issue, Aillaud et al. ( 6 ) and Nieuwenhuis et al. ( 7 ) report the identification and characterization of vasohibins as long-sought tubulin carboxypeptidases.

The “tubulin code” combines different α/β-tubulin isotypes with several post-translational modifications (PTMs) to generate microtubule diversity in cells. During cell division, specific microtubule populations in the mitotic spindle are differentially modified, but only recently, the functional significance of the tubulin code, with particular emphasis on the role specified by tubulin PTMs, started to be elucidated. This is the case of α-tubulin detyrosination, which was shown to guide chromosomes during congression to the metaphase plate and allow the discrimination of mitotic errors, whose correction is required to prevent chromosomal instability—a hallmark of human cancers implicated in tumor evolution and metastasis. Although alterations in the expression of certain tubulin isotypes and associated PTMs have been reported in human cancers, it remains unclear whether and how the tubulin code has any functional implications for cancer cell properties. Here, we review the role of the tubulin code in chromosome segregation during mitosis and how it impacts cancer cell properties. In this context, we discuss the existence of an emerging “cancer tubulin code” and the respective implications for diagnostic, prognostic and therapeutic purposes.

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end–directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E–dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E–driven chromosome congression is guided by microtubule detyrosination.

Chromosome congression during prometaphase culminates with the establishment of a metaphase plate, a hallmark of mitosis in metazoans. Classical views resulting from more than 100 years of research on this topic have attempted to explain chromosome congression based on the balance between opposing pulling and/or pushing forces that reach an equilibrium near the spindle equator. However, in mammalian cells, chromosome bi-orientation and force balance at kinetochores are not required for chromosome congression, whereas the mechanisms of chromosome congression are not necessarily involved in the maintenance of chromosome alignment after congression. Thus, chromosome congression and maintenance of alignment are determined by different principles. Moreover, it is now clear that not all chromosomes use the same mechanism for congressing to the spindle equator. Those chromosomes that are favorably positioned between both poles when the nuclear envelope breaks down use the so-called “direct congression” pathway in which chromosomes align after bi-orientation and the establishment of end-on kinetochore-microtubule attachments. This favors the balanced action of kinetochore pulling forces and polar ejection forces along chromosome arms that drive chromosome oscillatory movements during and after congression. The other pathway, which we call “peripheral congression”, is independent of end-on kinetochore microtubule-attachments and relies on the dominant and coordinated action of the kinetochore motors Dynein and Centromere Protein E (CENP-E) that mediate the lateral transport of peripheral chromosomes along microtubules, first towards the poles and subsequently towards the equator. How the opposite polarities of kinetochore motors are regulated in space and time to drive congression of peripheral chromosomes only now starts to be understood. This appears to be regulated by position-dependent phosphorylation of both Dynein and CENP-E and by spindle microtubule diversity by means of tubulin post-translational modifications. This so-called “tubulin code” might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed.

Cilia are evolutionarily conserved structures with many sensory and motility-related functions. The ciliary base, composed of the basal body and the transition zone, is critical for cilia assembly and function, but its contribution to cilia diversity remains unknown. Hence, we generated a high-resolution structural and biochemical atlas of the ciliary base of four functionally distinct neuronal and sperm cilia types within an organism, Drosophila melanogaster . We uncovered a common scaffold and diverse structures associated with different localization of 15 evolutionarily conserved components. Furthermore, CEP290 (also known as NPHP6) is involved in the formation of highly diverse transition zone links. In addition, the cartwheel components SAS6 and ANA2 (also known as STIL) have an underappreciated role in basal body elongation, which depends on BLD10 (also known as CEP135). The differential expression of these cartwheel components contributes to diversity in basal body length. Our results offer a plausible explanation to how mutations in conserved ciliary base components lead to tissue-specific diseases. Using electron and three-dimensional structured illumination microscopy methods, Jana et al. characterize the ciliary base in four different cilia types in Drosophila , discovering structural and protein component differences that may be linked to the diversified functions of cilia.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. Here, we identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Post-translational cycles of α-tubulin detyrosination and tyrosination generate microtubule diversity, the cellular functions of which remain largely unknown. Here we show that α-tubulin detyrosination regulates kinetochore-microtubule attachments to ensure normal chromosome oscillations and timely anaphase onset during mitosis. Remarkably, detyrosinated α-tubulin levels near kinetochore microtubule plus-ends depend on the direction of chromosome motion during metaphase. Proteomic analyses unveil that the KNL-1/MIS12/NDC80 (KMN) network that forms the core microtubule-binding site at kinetochores and the microtubule-rescue protein CLASP2 are enriched on tyrosinated and detyrosinated microtubules during mitosis, respectively. α-tubulin detyrosination enhances CLASP2 binding and NDC80 complex diffusion along the microtubule lattice in vitro. Rescue experiments overexpressing NDC80, including variants with slower microtubule diffusion, suggest a functional interplay with α-tubulin detyrosination for the establishment of a labile kinetochore-microtubule interface. These results offer a mechanistic explanation for how different detyrosinated α-tubulin levels near kinetochore microtubule plus-ends fine-tune load-bearing attachments to both growing and shrinking microtubules. Metaphase chromosomes oscillate while attached to growing and shrinking microtubules. Here, the authors show that an α-tubulin detyrosination gradient on kinetochore microtubules fine-tunes load-bearing attachments during chromosome oscillations.

Multipolar spindles are a feature of cancer cells often associated with chromosomal aberrations. In the final Review in our Series on Genomic Instability, Logarinho and Maiato discuss how multipolar spindles form, with an emphasis on the role of the loss of spindle pole integrity in this process. Mitotic spindle bipolarity is essential for faithful segregation of chromosomes during cell division. Multipolar spindles are often seen in human cancers and are usually associated with supernumerary centrosomes that result from centrosome overduplication or cytokinesis failure. A less-understood path to multipolar spindle formation may arise due to loss of spindle pole integrity in response to spindle and/or chromosomal forces. Here we discuss the different routes leading to multipolar spindle formation, focusing on spindle multipolarity without centrosome amplification. We also present the distinct and common features between these pathways and discuss their therapeutic implications.