Marin Barisic

Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

Human Spindly is required for kinetochore localization of cytoplasmic dynein, which is essential for poleward movement of chromosomes and for kinetochore protein streaming. In addition, Spindly controls the activity and kinetochore abundance of the RZZ complex, which contributes to microtubule attachment and mitotic checkpoint activity.

Microtubule detyrosination guides chromosomes during mitosis

Chromosomes: Let me be your guide The correct alignment of chromosomes at the center of the mitotic spindle—the metaphase plate—before cell division is one of the key mechanisms for the maintenance of genomic stability. But is there anything special about the microtubules of the spindle that helps this process? Barisic et al. demonstrate that chromosome alignment at the cell equator is controlled by a specific posttranslational modification of selected microtubules oriented toward the center of the mitotic spindle. Science, this issue p. 799 Microtubule detyrosination works as a navigation system for kinetochore-based chromosome motility during cell division. 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.

Human chromokinesins promote chromosome congression and spindle microtubule dynamics during mitosis

Human chromokinesins hKID and KIF4A contribute to proper attachment of chromosomes by controlling the positioning of the chromosome arms and microtubule dynamics, respectively.

CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment

Loss of spindle-pole integrity during mitosis leads to multipolarity independent of centrosome amplification. Multipolar-spindle conformation favours incorrect kinetochore–microtubule attachments, compromising faithful chromosome segregation and daughter-cell viability. Spindle-pole organization influences and is influenced by kinetochore activity, but the molecular nature behind this critical force balance is unknown. CLASPs are microtubule-, kinetochore- and centrosome-associated proteins whose functional perturbation leads to three main spindle abnormalities: monopolarity, short spindles and multipolarity. The first two reflect a role at the kinetochore–microtubule interface through interaction with specific kinetochore partners, but how CLASPs prevent spindle multipolarity remains unclear. Here we found that human CLASPs ensure spindle-pole integrity after bipolarization in response to CENP-E- and Kid-mediated forces from misaligned chromosomes. This function is independent of end-on kinetochore–microtubule attachments and involves the recruitment of ninein to residual pericentriolar satellites. Distinctively, multipolarity arising through this mechanism often persists through anaphase. We propose that CLASPs and ninein confer spindle-pole resistance to traction forces exerted during chromosome congression, thereby preventing irreversible spindle multipolarity and aneuploidy.

Cdk1 and Plk1 mediate a CLASP2 phospho-switch that stabilizes kinetochore–microtubule attachments

Cdk1 phosphorylation of CLASP2 promotes Plk1 recruitment to kinetochores and is required for stabilization of kinetochore–microtubule attachments, chromosome alignment, and satisfaction of the spindle assembly checkpoint.

Cyclin-Dependent Kinase 16/PCTAIRE Kinase 1 Is Activated by Cyclin Y and Is Essential for Spermatogenesis

ABSTRACT Cyclin-dependent kinase 16 (CDK16, PCTK1) is a poorly characterized protein kinase, highly expressed in the testis and the brain. Here, we report that CDK16 is activated by membrane-associated cyclin Y (CCNY). Treatment of transfected human cells with the protein kinase A (PKA) activator forskolin blocked, while kinase inhibition promoted, CCNY-dependent targeting of CDK16-green fluorescent protein (GFP) to the cell membrane. CCNY binding to CDK16 required a region upstream of the kinase domain and was found to be inhibited by phosphorylation of serine 153, a potential PKA phosphorylation site. Thus, in contrast to other CDKs, CDK16 is regulated by phosphorylation-controlled cyclin binding. CDK16 isolated from murine testis was unphosphorylated, interacted with CCNY, and exhibited kinase activity. To investigate the function of CDK16 in vivo, we established a conditional knockout allele. Mice lacking CDK16 developed normally, but male mice were infertile. Spermatozoa isolated from their epididymis displayed thinning and elongation of the annulus region, adopted a bent shape, and showed impaired motility. Moreover, CDK16-deficient spermatozoa had malformed heads and excess residual cytoplasm, suggesting a role of CDK16 in spermiation. Thus, CDK16 is a membrane-targeted CDK essential for spermatogenesis.

Kinetochore motors drive congression of peripheral polar chromosomes by overcoming random arm-ejection forces

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. These proteins include the minus-end-directed kinetochore motor dynein, and the plus-end-directed motors CENP-E at kinetochores and chromokinesins on chromosome arms. 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.

Mechanisms of Chromosome Congression during Mitosis

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.

The Tubulin Code: A Navigation System for Chromosomes during Mitosis

Before chromosomes segregate during mitosis in metazoans, they align at the cell equator by a process known as chromosome congression. This is in part mediated by the coordinated activities of kinetochore motors with opposite directional preferences that transport peripheral chromosomes along distinct spindle microtubule populations. Because spindle microtubules are all made from the same α/β-tubulin heterodimers, a critical longstanding question has been how chromosomes are guided to specific locations during mitosis. This implies the existence of spatial cues/signals on specific spindle microtubules that are read by kinetochore motors on chromosomes and ultimately indicate the way towards the equator. Here, we discuss the emerging concept that tubulin post-translational modifications (PTMs), as part of the so-called tubulin code, work as a navigation system for kinetochore-based chromosome motility during early mitosis.

Spindly switch controls anaphase: Spindly and RZZ functions in chromosome attachment and mitotic checkpoint control

Spindly is a coiled-coil domain containing protein, also known as CCDC99, that is an important regulator of the mitotic checkpoint. It belongs to a functionally conserved family of proteins that controls the localization and function of cytoplasmic dynein at kinetochores. At kinetochores Spindly interacts with the RZZ (Rod/ZW10/Zwilch) complex and is required for the recruitment of cytoplasmic dynein to kinetochores. Loss of Spindly results in loss of kinetochore dynein functions, which impairs poleward chromosome movements and mitotic checkpoint inactivation. In addition, Spindly regulates the establishment of stable microtubule attachments via regulation of the RZZ complex. Thus, Spindly couples dynein-mediated poleward chromosome movements to the establishment of stable microtubule attachments and subsequent mitotic checkpoint inactivation and provides an important switch for the control of mitosis.