Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

Good, Matthew C.; Vahey, Michael D.; Skandarajah, Arunan; Fletcher, Daniel A.; Heald, Rebecca

doi:10.1126/science.1243147

Cited by 240 publications

(308 citation statements)

References 37 publications

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“…Most intriguingly, the spindle size scales linearly with the cell size up to a certain limit; and such a biphasic scaling relationship, as well as the critical cell size and spindle size for the biphasic transition, appear surprisingly consistent across metazoan species (5,8,10). Such size scaling is also partially reproduced in in vitro spindle reconstitution experiments (15,16). From the physics point of view, such universal size-scaling phenomena are highly significant, as they suggest intrinsic principles in the spatial organization of the mitotic spindle.…”

Section: Introductionmentioning

confidence: 75%

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint

Chen

Liu

2016

Biophysical Journal

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Chromosome segregation during mitosis hinges on proper assembly of the microtubule spindle that establishes bipolar attachment to each chromosome. Experiments demonstrate allometry of mitotic spindles and a universal scaling relationship between spindle size and cell size across metazoans, which indicates a conserved principle of spindle assembly at play during evolution. However, the nature of this principle is currently unknown. Researchers have focused on deriving the mechanistic underpinning of the size scaling from the mechanical aspects of the spindle assembly process. In this work we take a different standpoint and ask: What is the size scaling for? We address this question from the functional perspectives of spindle assembly checkpoint (SAC). SAC is the critical surveillance mechanism that prevents premature chromosome segregation in the presence of unattached or misattached chromosomes. The SAC signal gets silenced after and only after the last chromosome-spindle attachment in mitosis. We previously established a model that explains the robustness of SAC silencing based on spindle-mediated spatiotemporal regulation of SAC proteins. Here, we refine the previous model, and find that robust and timely SAC silencing entails proper size scaling of mitotic spindle. This finding provides, to our knowledge, a novel, function-oriented angle toward understanding the observed spindle allometry, and the universal scaling relationship between spindle size and cell size in metazoans. In a broad sense, the functional requirement of robust SAC silencing could have helped shape the spindle assembly mechanism in evolution.

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Section: Introductionmentioning

confidence: 75%

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint

Chen

Liu

2016

Biophysical Journal

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“…This characteristic mitotic cell shape provides a geometrical boundary that both restricts and directs spindle size and orientation 35,36 . Microfluidic techniques, provide a level of geometrical confinement that accurately resembles the situation observed in living cells and therefore permits the bottom-up reconstruction of mitotic spindles in vitro.…”

Section: Representative Resultsmentioning

confidence: 99%

“…In addition, growing into a physical barrier can induce microtubule catastrophes. Also, spindle assembly within a confined geometry can lead to a gradual depletion of individual components, such as tubulin, which in turn directly affects the microtubule growth rate 36 . These are all essential determinants of spindle assembly, which can be studied using the assays described here.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Vleugel

Roth

Groenendijk

et al. 2016

JoVE

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Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

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“…Changing spindle size in developing embryos had no effect on chromosome segregation, but interfered with spindle orientation, suggesting that it is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization. This idea was further explored via an innovative system, in which Xenopus egg extracts were encapsulated using microfluidic technology (99,100). Both studies beautifully demonstrated that reductions in cytoplasmic volume, rather than developmental cues or changes in cell shape, were sufficient to recapitulate spindle scaling observed in Xenopus embryos.…”

Section: Assembly and Scaling Of Self-organized Microtubule Structuresmentioning

confidence: 99%

Building the Microtubule Cytoskeleton Piece by Piece

Alfaro-Aco

Petry

2015

Journal of Biological Chemistry

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The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of selforganized MT networks in the cell. The microtubule (MT)2 cytoskeleton forms the skeletal framework that gives eukaryotic cells their shape and organizes their cytoplasm by positioning organelles, providing tracks for transport, and establishing cell polarity. In an interphase cell, the MT cytoskeleton is also critical for cell motility and a key constituent of cilia and flagella. During cell division, the MT cytoskeleton gets remodeled into a spindle structure that segregates chromosomes. Each of these functions relies on a specific MT architecture, which must be capable of rapid and prolonged change followed by an eventual resumption of a steady state to respond to the cellular environment and morphology changes during growth and differentiation.MTs are made of ␣/␤-tubulin heterodimers, which assemble into a polar, cylindrical structure in the presence of GTP and above the so-called critical concentration in vitro. MT growth phases alternate with swift shrinkage phases (dynamic instability), and their transitions are referred to as catastrophe (switching from growth to shrinkage) and rescue (switching from shrinkage to growth) (1). In cells, a plethora of different MT-associated proteins (MAPs) regulate the MT-inherent abilities of MT nucleation and dynamics ( Fig. 1A) (2). In addition, MT cross-linking proteins connect MTs into networks and molecular motors use MTs as tracks for cargo transport or transport MTs themselves (Fig. 1A). Altogether, different combinations of these four basic groups of MAP activities drive the self-organization of the MT cytoskeleton into discrete three-dimensional patterns (Fig. 1B) (3). Thus, they establish, maintain, and disassemble functional MT structures that are observed on the cellular level.Traditionally, individual MAPs were identified by loss-offunction experiments in cells followed by their detailed in vivo and in vitro characterization. During the past decade, highthroughput genomic and proteomic screens accelerated MAP discovery by cataloging RNAi phenotypes and identifying novel microtubule binders, resulting in comprehensive lists of candidates involved in organizing the MT cytoskeleton in various cell states (4 -7). Now, the challenge is to understand how these MAPs work together to establish the physiological MT architecture of the cell. What specific MAP building blocks can generate the MT networks that shape a dendrite or a polarized epithelial cell (...

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Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

Cited by 240 publications

References 37 publications

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Building the Microtubule Cytoskeleton Piece by Piece

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