Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Vleugel, Mathijs; Roth, Sophie; Groenendijk, Celebrity F.; Dogterom, Marileen

doi:10.3791/54278

Cited by 20 publications

(17 citation statements)

References 42 publications

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“…Quantitative force-balance models were later applied to Drosophila spindle assembly (28)(29)(30)(31), and the same ideas have more recently been extended to human cells (32). In parallel with work on spindles, force-balance ideas have been studied in reconstituted MTmotor systems (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45).…”

Section: Introductionmentioning

confidence: 99%

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Betterton

Blackwell

Edelmaier

et al. 2017

Biophysical Journal

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Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition; this is the most fundamental construction problem that the spindle must solve. Microtubules, motors, and cross-linkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown. We describe a physical model that exhibits de novo bipolar spindle formation. We began with physical properties of fission-yeast spindle pole body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive cross-linkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective selfassembly. By varying the features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. When kinesin-5 motors are present, their bidirectionality is essential, but spindles can form in the presence of passive cross-linkers alone. We also identify characteristic failed states of spindle assemblythe persistent monopole, X spindle, separated asters, and short spindle, which are avoided by the creation and maintenance of antiparallel microtubule overlaps. Our model can guide the identification of new, multifaceted strategies to induce mitotic catastrophes; these would constitute novel strategies for cancer chemotherapy.

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Section: Introductionmentioning

confidence: 99%

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Betterton

Blackwell

Edelmaier

et al. 2017

Biophysical Journal

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“…For example, NTA-lipid has been widely used to induce recruitment of His-tagged proteins to one leaflet of liposomes and supported lipid bilayers (Gizeli & Glad, 2004;Stachowiak et al, 2012). Additionally, biotinylated lipids have been used to recruit oligomeric assemblies of streptavidin and biotinylated proteins to the boundary of water-in-oil emulsions (Vleugel, Roth, Groenendijk, & Dogterom, 2016). Covalent chemistry can be utilized to overcome weak protein-lipid interactions.…”

Section: Expanded Toolkit For Protein Recruitment To Compartment Bomentioning

confidence: 99%

Probing the biology of cell boundary conditions through confinement of Xenopus cell‐free cytoplasmic extracts

Bermudez

Chen

Einstein

et al. 2017

Genesis

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Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.

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“…A variety of dynamic processes occur in the spindle, including MTs undergoing dynamic instability (A, green and red arrows represent growing and shrinking MTs); poleward MT flux (B, yellow arrows); chromosome movements (C); motor-driven antiparallel MT sliding (D); dynein dependent, minus-end directed MT transport (E); and orientational movements of the spindle poles (F) (reproduced with permission from Wittmann et al 46 ). (b) Flow focusing microfluidic chip, as described in 32, 33 , used to generate W/O emulsion droplets with a diameter in the range 12-25μm. Top: Flow focusing junction.…”

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confidence: 99%

Reconstitution of basic mitotic spindles in cell-like confinement

Roth

Gârlea

Vleugel

et al. 2019

Preprint

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Bipolar organization of the mitotic spindle is the result of forces generated by dynamic microtubules and associated proteins in interaction with chromosomes and the cell boundary1–4. Biophysical experiments on isolated spindle components have provided important insights into the force-generating properties of different components5–8, but a quantitative understanding of the force balance that results from their concerted action is lacking. Here we present an experimental platform based on water-in-oil emulsion droplets that allows for the bottom-up reconstitution of basic spindles. We find a typical metaphase organization, where two microtubule asters position symmetrically at moderate distance from the mid-zone, is readily obtained even in the absence of chromosomes. Consistent with simulations, we observe an intrinsic repulsive force between two asters that can be counterbalanced alternatively by cortical pulling forces, anti-parallel microtubule crosslinking, or adjustment of microtubule dynamics, emphasizing the robustness of the system. Adding motor proteins that slide anti-parallel microtubules apart drives the asters to maximum separation, as observed in cells during anaphase9,10. Our platform offers a valuable complementary approach to in vivo experiments where essential mitotic components are typically removed, instead of added, one by one.

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Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Cited by 20 publications

References 42 publications

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

Probing the biology of cell boundary conditions through confinement of Xenopus cell‐free cytoplasmic extracts

Reconstitution of basic mitotic spindles in cell-like confinement

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