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

Summary

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.

Abstract

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.

Introduction

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 vitro^1,2. 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 segregation^1,2,

Protocol

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^21,22.
   2. Measure the concentrations of fragments using a fluorospectrometer. Combine 100 ng o....

Results

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 (

Discussion

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.......

Materials

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