Cancer is a group of diseases in which cells divide continuously and excessively. Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms, to ensure the production of two genetically identical cells. Cell cycle checkpoints operate as DNA surveillance mechanisms that prevent the accumulation and propagation of genetic errors during cell division. Checkpoints can delay cell cycle progression or, in response to irreparable DNA damage, induce cell cycle exit or cell death. Cancer-associated mutations that perturb cell cycle control allow continuous cell division chiefly by compromising the ability of cells to exit the cell cycle. Continuous rounds of division, however, create increased reliance on other cell cycle control mechanisms to prevent catastrophic levels of damage and maintain cell viability. New detailed insights into cell cycle control mechanisms and their role in cancer reveal how these dependencies can be best exploited in cancer treatment.

Cell cycle control in cancer.

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.U-ExM enables near-native expansion microscopy of samples in vitro and in cells. The combination of U-ExM with confocal microscopy and HyVolution revealed details of centriole chirality that were previously accessible only by electron microscopy.

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

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.

https://www.mdpi.com/2079-7737/6/1/13/pdf

Mechanisms of Chromosome Congression during Mitosis

Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.

/pdf/a-liquid-like-spindle-domain-promotes-acentrosomal-spindle-2ec9cclog0.pdf

A liquid-like spindle domain promotes acentrosomal spindle assembly in mammalian oocytes

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes.

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term ‘bridging fibre’, bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

/pdf/overlap-microtubules-link-sister-k-fibres-and-balance-the-1admaeoxwc.pdf

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

What forces drive chromosome segregation remains one of the most challenging questions in cell division. Even though the duration of anaphase is short, it is of utmost importance for genome fidelity that no mistakes are made. Seminal studies in model organisms have revealed different mechanisms operating during chromosome segregation in anaphase, but the translation of these mechanisms to human cells is not straightforward. Recent work has shown that kinetochore fiber depolymerization during anaphase A is largely motor independent, whereas spindle elongation during anaphase B is coupled to sliding of interpolar microtubules in human cells. In this Review, we discuss the current knowledge on the mechanisms of force generation by kinetochore, interpolar and astral microtubules. By combining results from numerous studies, we propose a comprehensive picture of the role of individual force-producing and -regulating proteins. Finally, by linking key concepts of anaphase to most recent data, we summarize the contribution of all proposed mechanisms to chromosome segregation and argue that sliding of interpolar microtubules and depolymerization at the kinetochore are the main drivers of chromosome segregation during early anaphase in human cells.

Force-generating mechanisms of anaphase in human cells

Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation.

The mitotic spindle is a highly organized and dynamic structure required for segregation of the genetic material into two daughter cells. Although most of the individual players involved in building the spindle have been characterized in vitro, a general understanding of how all of the spindle players act together in vivo is still missing. Hence, in recent years, experiments have focused on introducing mechanical perturbations of the spindle on a micron scale, thereby providing insight into its function and organization, as well as into forces acting in the spindle. Among different types of mechanical perturbations, optical ones are more flexible, less invasive, and more precise than other approaches. In this chapter, we describe a detailed protocol for cutting the microtubule bundles in human cells using a near-infrared femtosecond laser. This type of laser microsurgery provides the ability to precisely sever a single microtubule bundle while preserving spindle integrity and dynamics. Furthermore, we describe quantitative measurements obtained from the response of a severed microtubule bundle to laser ablation, which reveal the structure and function of individual parts of the spindle, such as the bridging fiber connecting sister k-fibers. Finally, the method described here can be easily combined with other quantitative techniques to address the complexity of the spindle.

Kruno Vukušić

Papers

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes.

Force-generating mechanisms of anaphase in human cells

Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation.

Dissection and characterization of microtubule bundles in the mitotic spindle using femtosecond laser ablation.