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Biochemistry

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Published: September 27th, 2018

DOI:

10.3791/58240

1Department of Biophysics, University of Michigan, Ann Arbor, 2Department of Chemistry, University of Michigan, Ann Arbor, 3Department of Computer Science, Wayne State University, 4Department of Physics, University of Michigan, Ann Arbor

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Cytoplasmic extracts prepared from Xenopus laevis eggs represent one of the most predominant models for the biochemical study of cell cycles, given the large volume of oocytes, the rapid cell cycle progression, and the capability of reconstituting mitotic events in vitro1,2. This system has allowed the initial discovery and mechanistic characterization of essential cell-cycle regulators like maturation-promoting factor (MPF) as well as downstream mitotic processes including spindle assembly and chromosome segregation1,2,

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All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of University of Michigan.

1. Preparation of Materials for Cell Cycle Reconstitution and Detection

  1. Gibson Assembly cloning for the plasmid DNA construction and mRNA purification of securin-mCherry
    1. Prepare three DNA fragments including a pMTB2 vector backbone, securin, and mCherry through polymerase chain reaction (PCR) and gel purification

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In Figure 2, we show that this protocol produces mitotic oscillations in both simple, nuclear-free cells as well as complicated cells with nuclei, where the oscillator drives the cyclic alternation of nuclei formation and deformation. The nuclei-free droplets generate mitotic oscillations up to 30 undamped cycles over the time span of 92 hours, as indicated by the periodic synthesis and degradation of a fluorescence reporter securin-mCherry (

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We have presented a novel method for developing a high-throughput artificial cell system that enables in vitro reconstitution and long-term tracking of self-sustained cell-cycle oscillations at the single-cell level. There are several critical steps that make this method successful. First, freshly squeezed Xenopus eggs with a good quality, compared with laid eggs, tend to produce extracts with longer-lasting oscillation activity. Second, encapsulation of extracts within the surfactant-stabilized microen.......

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We thank Madeleine Lu for constructing securin-mCherry plasmid, Lap Man Lee, Kenneth Ho and Allen P Liu for discussions about droplet generation, Jeremy B. Chang and James E. Ferrell Jr for providing GFP-NLS construct. This work was supported by the National Science Foundation (Early CAREER Grant #1553031), the National Institutes of Health (MIRA #GM119688), and a Sloan Research Fellowship.

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Name Company Catalog Number Comments
Xenopus laevis frogs Xenopus-I Inc.
QIAprep spin miniprep kit QIAGEN 27104
QIAquick PCR Purification Kit (250) QIAGEN 28106
mMESSAGE mMACHINE SP6 Transcription Kit Ambion AM1340
BL21 (DE3)-T-1 competent cell Sigma-Aldrich B2935
Calcium ionophore Sigma-Aldrich A23187
Hoechst 33342 Sigma-Aldrich B2261 Toxic
Trichloro Sigma-Aldrich 448931 Toxic
(1H,1H,2H,2H-perfluorooctyl) silane
PFPE-PEG surfactant Ran Biotechnologies 008-FluoroSurfactant-2wtH-50G
GE Healthcare Glutathione Sepharose 4B beads Sigma-Aldrich GE17-0756-01
PD-10 column Sigma-Aldrich GE17-0851-01
VitroCom miniature hollow glass tubing VitroCom 5012
Olympus SZ61 Stereo Microscope Olympus
Olympus IX83 microscope Olympus
Olympus FV1200 confocal microscope Olympus
NanoDrop spectrophotometer Thermofisher ND-2000
0.4 mL Snap-Cap Microtubes E&K Scientific 485050-B
 PureLink RNA Mini Kit ThermoFisher(Ambion) 12183018A
Fisherbrand Analog Vortex Mixer Fisher Scientific 2215365
Imaris Bitplane Version 7.3 Image analysis software

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