Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

Nixon, Faye M; Honnor, Thomas R.; Clarke, Nicholas I.; Starling, Georgina P.; Beckett, Alison J.; Johansen, Adam M.; Brettschneider, Julia; Prior, Ian A.; Royle, Stephen

doi:10.1242/jcs.203877

Cited by 40 publications

(38 citation statements)

References 30 publications

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“…S3 B). Thus, not all MTs of the metaphase spindle body extend to the poles, consistent with previous reports (Kamasaki et al, 2013;Nixon et al, 2017).…”

Section: Augmin-dependent Mannersupporting

confidence: 92%

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

David

Roudot

Legant

et al. 2018

Preprint

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Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatidsto each nascent daughter cell. Early spindle assembly models postulated that spindle-pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at non-centrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus-ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubules. By ultra-fast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth towards individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore-fiber assembly.This normalization enables to integrate thousands of dROI across space, time and acquisitions. The probability distributions, ( , ), were then computed for different time intervals relative to nuclear envelope disassembly as shown in Fig. 5.

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“…S3 B). Thus, not all MTs of the metaphase spindle body extend to the poles, consistent with previous reports (Kamasaki et al, 2013;Nixon et al, 2017).…”

Section: Augmin-dependent Mannersupporting

confidence: 92%

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

David

Roudot

Legant

et al. 2018

Preprint

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“…It has been previously reported that non-kinetochore MTs could extend along the k-fiber to the region between sister kinetochores in metaphase spindles of human cells (McIntosh and Landis, 1971;Nixon et al, 2017). We found that almost all of these nonkinetochore MT bundles act as a bridge between sister k-fibers (Kajtez et al, 2016;Polak et al, 2017) (Fig.…”

Section: Coupling Of Depolymerizing Mts To Spindle Poles and Kinetochsupporting

confidence: 57%

Force-generating mechanisms of anaphase in human cells

Vukušić

Buđa

Tolić

2019

Journal of Cell Science

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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.

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“…We also observed multiple kinetochore/lateral MT bundles converging into larger MT assemblies ( Figure 9A The presence of associations between k-fibers of 9-15 MTs and large lateral MT bundles (comprising 9-34 MTs) seems to be peculiar to Drosophila S2 cells. MT bundles running laterally to kinetochores have been previously observed by TEM in both longitudinal and cross-sections of several spindle types including those of rat kangaroo PTK cells (Brinkley and Cartwright, 1971;McDonald et al 1992) and HeLa cells (Wendell et al, 1993;Booth et al, 2011;Nixon et al 2017), as well as in spindles assembled from Xenopus egg extracts (Ohi et al, 2003). However, in all cases, in contrast to S2 cells, the lateral MT bundles appeared to contain much fewer MTs than the kinetochore fibers.…”

Section: Analysis Of Longitudinal and Transverse Serial Sections (mentioning

confidence: 64%

Ultrastructural analysis of intracellular membrane and microtubule behavior during mitosis ofDrosophilaS2 cells

Strunov

Boldyreva

Andreyeva

et al. 2017

Preprint

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S2 cells are one of the most widely used Drosophila melanogaster cell lines for molecular dissection of mitosis using RNA interference (RNAi). However, a detailed and complete description of S2 cell mitosis at the ultrastructural level is still missing.Here, we analyzed by transmission electron microscopy (TEM) a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behavior. This unbiased approach allowed us to discover that S2 cells exhibit a characteristic behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of ER membranes that associate with mitochondria preventing their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached kfibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. In summary, we describe several previously unknown features of membrane and microtubule organization during S2 cell mitosis. The genetic determinants of these mitotic features of can now be investigated using an RNAi-based approach, which is particularly easy and efficient in S2 cells List of abbreviations:DNM, double nuclear membrane; ER, endoplasmic reticulum; MT, microtubule; NEBD, nuclear envelope breakdown; NPC, nuclear pore complex; QNM, quadruple nuclear membrane; RDM, residual double membrane; SE, spindle envelope; TEM, transmission electron microscopy; not peer-reviewed)

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Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

Cited by 40 publications

References 30 publications

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

Augmin-mediated amplification of long-lived spindle microtubules directs plus-ends to kinetochores

Force-generating mechanisms of anaphase in human cells

Ultrastructural analysis of intracellular membrane and microtubule behavior during mitosis ofDrosophilaS2 cells

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