Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells

Pereira, António J.; Matos, Irina; Lince-Faria, Mariana; Maiato, Hélder

doi:10.1007/978-1-60327-993-2_9

Cited by 19 publications

(11 citation statements)

References 19 publications

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“…The inclusion of a kinetochore marker in this study and the observed variability of inter-kinetochore distance after k-fiber cut explains previous observations in which no detectable kinetochore relaxation was observed without the use of a kinetochore marker [29]. Laser microsurgery was performed essentially as described in [373]. …”

Section: Figuresupporting

confidence: 52%

Mechanisms of Chromosome Congression during Mitosis

Maiato

Gomes

Sousa

et al. 2017

Biology

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161

176

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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.

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Section: Figuresupporting

confidence: 52%

Mechanisms of Chromosome Congression during Mitosis

Maiato

Gomes

Sousa

et al. 2017

Biology

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161

176

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“…mNeonGreen-PICH cells were imaged every 60s at 4 z-sections at 1 μm intervals. The laser microsurgery was conducted as described previously (Pereira et al, 2009) and is detailed in the Supplemental Experimental Procedures. An extended description of the analysis of the time-lapse movies and kinetochore motion is also included in the Supplemental Experimental Procedures.…”

Section: Methodsmentioning

confidence: 99%

A Regulatory Switch Alters Chromosome Motions at the Metaphase-to-Anaphase Transition

Barry

Schweizer

et al. 2016

Cell Reports

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SummaryTo achieve chromosome segregation during mitosis, sister chromatids must undergo a dramatic change in their behavior to switch from balanced oscillations at the metaphase plate to directed poleward motion during anaphase. However, the factors that alter chromosome behavior at the metaphase-to-anaphase transition remain incompletely understood. Here, we perform time-lapse imaging to analyze anaphase chromosome dynamics in human cells. Using multiple directed biochemical, genetic, and physical perturbations, our results demonstrate that differences in the global phosphorylation states between metaphase and anaphase are the major determinant of chromosome motion dynamics. Indeed, causing a mitotic phosphorylation state to persist into anaphase produces dramatic metaphase-like oscillations. These induced oscillations depend on both kinetochore-derived and polar ejection forces that oppose poleward motion. Thus, our analysis of anaphase chromosome motion reveals that dephosphorylation of multiple mitotic substrates is required to suppress metaphase chromosome oscillatory motions and achieve directed poleward motion for successful chromosome segregation.

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“…Examples of mechanical approaches to improve live cell imaging include: coating coverslips (e.g. with Poly-L-Lysine) to help keep dividing cells flat; confining dividing cells in PDMS devices of different heights (Le Berre, Aubertin and Piel, 2012); and laying an agar pad on top of a cell to reduce mitotic rounding and movement (Fukui, Yumura and Yumura 1987; Pereira, Matos, Lince-Faria and Maiato, 2009). Examples of physical perturbation approaches include microneedles to exert and measure tension on individual chromosomes and kinetochores in insect spermatocyte cells (Nicklas, 1983; Nicklas and Staehly, 1967); optical tweezers to move chromosomes inside mammalian cells (Liang, Wright, He and Berns, 1991); and laser ablation to probe kinetochore motility (Khodjakov and Rieder, 1996), kinetochore signaling (Rieder, Cole, Khodjakov and Sluder, 1995) and spindle mechanics (Snyder, Armstrong, Stonington, Spurck and Pickett-Heaps, 1991).…”

Section: Spindle Compression To Image and Perturb Kinetochoresmentioning

confidence: 99%

Imaging and physically probing kinetochores in live dividing cells

Kuhn

Dumont

2014

Methods in Cell Biology

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The kinetochore mediates chromosome segregation at cell division. It is the macromolecular machine that links chromosomes to spindle microtubules, and is made of more than 100 protein species in mammalian cells. Molecular tools are presently revealing the biochemical interactions and regulatory mechanisms that ensure proper kinetochore function. Here, we discuss two approaches for imaging and physically probing kinetochores despite mitotic cell rounding and rapid kinetochore dynamics. First, we describe how mild spindle compression can improve kinetochore imaging, and how stronger compression can mechanically perturb the spindle and kinetochores. Second, we describe how simultaneously imaging two-colored kinetochore reporter probes at sub-pixel resolution can report on kinetochore structural dynamics under cellular forces. We hope that the experimental details we provide here will make these two approaches broadly accessible and help move forward our understanding of kinetochore function – and make these approaches adaptable to the study of other cellular structures.

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Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells

Cited by 19 publications

References 19 publications

Mechanisms of Chromosome Congression during Mitosis

Mechanisms of Chromosome Congression during Mitosis

A Regulatory Switch Alters Chromosome Motions at the Metaphase-to-Anaphase Transition

Imaging and physically probing kinetochores in live dividing cells

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