We thank Madeleine Lu for constructing securin-mCherry plasmid, Lap Man Lee,&#160;Kenneth Ho and Allen P Liu&#160;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.

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

<ol>
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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...

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 segregation1,2,3,4,5,6,7,8,9,10,11. The Xenopus egg extracts have also been used for detailed dissection of the regulatory networks of the cell cycle clock8,12,13,14 and for studies of the DNA damage/replication checkpoint15 and the mitotic spindle assembly checkpoint16,17,18.
These studies of cell cycles using the Xenopus egg extracts have mainly been based on bulk measurements. However, conventional bulk reaction assays may not mimic real cell behaviors, given a major discrepancy in their dimensions and subcellular spatial compartmentalization of reaction molecules. Moreover, bulk measurements of mitotic activities are prone to giving a limited number of cycles before quickly damping8. These disadvantages of bulk reactions have prevented the extract system to provide further understanding of complex clock dynamic properties and functions. Recent studies have encapsulated cell-free cytostatic factor-arrested (CSF) Xenopus extracts19,20 into size-defined cell-like compartments, which have helped elucidate how spindle size is modulated by the cytoplasmic volume. However, this in vitro system is arrested at metaphase of meiosis II by the action of cytostatic factor1, and a system capable of long-term sustained oscillations at the single-cell level is needed for further investigation of the cell cycle oscillator.
To study cell cycle oscillations with single-cell resolution, we have developed a cell-scale, high-throughput system for reconstitution and simultaneous measurement of multiple self-sustained mitotic oscillatory processes in individual microemulsion droplets. In this detailed video protocol, we demonstrate the creation of the artificial mitotic oscillation system by encapsulating cycling Xenopus laevis egg cytoplasm in microemulsions of sizes ranging from 10 to 300 &#181;m. In this system, mitotic oscillations including chromosome condensation and de-condensation, nuclear envelope breakdown and reformation, and the degradation and synthesis of anaphase substrates (e.g., securin-mCherry in this protocol) were successfully reconstituted.

<table border="0" cellpadding="0" cellspacing="0" width="593">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Xenopus laevis frogs</td>
			<td style="width:93px;">Xenopus-I Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">QIAprep spin miniprep kit</td>
			<td style="width:93px;">QIAGEN</td>
			<td style="width:119px;">27104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">QIAquick PCR Purification Kit (250)</td>
			<td style="width:93px;">QIAGEN</td>
			<td style="width:119px;">28106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">BL21 (DE3)-T-1 competent cell</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">B2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Calcium ionophore</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">A23187</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Hoechst 33342</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">B2261</td>
			<td style="width:167px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trichloro</td>
			<td rowspan="2" style="width:93px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">448931</td>
			<td rowspan="2" style="width:167px;">Toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">(1H,1H,2H,2H-perfluorooctyl) silane</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:215px;">PFPE-PEG surfactant</td>
			<td style="width:93px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant-2wtH-50G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">GE Healthcare Glutathione Sepharose 4B beads</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">GE17-0756-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">PD-10 column</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">GE17-0851-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">VitroCom miniature hollow glass tubing</td>
			<td style="width:93px;">VitroCom</td>
			<td style="width:119px;">5012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Olympus SZ61 Stereo Microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Olympus IX83 microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">Olympus FV1200 confocal microscope</td>
			<td style="width:93px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">NanoDrop spectrophotometer</td>
			<td style="width:93px;">Thermofisher</td>
			<td style="width:119px;">ND-2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:215px;">0.4 mL Snap-Cap Microtubes</td>
			<td style="width:93px;">E&#38;K Scientific</td>
			<td style="width:119px;">485050-B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="54" rowspan="2" style="height:55px;width:215px;">&#160;PureLink RNA Mini Kit</td>
			<td rowspan="2" style="width:93px;">ThermoFisher&#65288;Ambion&#65289;</td>
			<td rowspan="2" style="width:119px;">12183018A</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Fisherbrand Analog Vortex Mixer</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">2215365</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Imaris</td>
			<td style="width:93px;">Bitplane</td>
			<td style="width:119px;">Version 7.3</td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

reconstitution of cell-cycle oscillations in microemulsions of cell-free xenopus egg extracts

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.

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 (<strong class="xf...

Watch this Scientific Journal Video about Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts at JoVE.com

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

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.

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

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

This method can help answer key questions in the system biology field, such as the regulating mechanism behind the mitotic network and the dynamic properties and functions of the cell cycle clock. The main advantage of this technique is that it will generate a large quantity of droplets with multiple cell cycles in each, providing us with a high-throughput framework for quantitative manipulation and analysis. To begin, discard batches of eggs that have unclear boundaries between animal and vegetal poles or irregular white spots on top of animal poles.Next, select an appropriate batch of eggs from one female frog, and transfer the eggs to a 600-milliliter beaker. Pour out an excess of 0.2x MMR buffer, and gently add cysteine in 1x extract buffer to the eggs. After this, shake the beaker vigorously by hand to remove the jelly coats of the eggs.Repeat this wash three times with a total of 800 milliliters of cysteine buffer or until the eggs aggregate and settle at the bottom of the beaker. After the jelly coat is removed, wash the eggs four times in one liter of 0.2x MMR solution. Use a glass pipette to pick up and discard the eggs that turn white.Pour the MMR buffer out of the beaker, and add 200 milliliters of calcium ionophore solution into the eggs. Use a glass pipette to stir the eggs gently until they are activated, and remove the calcium ionophore solution. Leaving the eggs in calcium ionophore solution for too long will initiate apoptosis.We stir the eggs for 1.5 minutes and check the activation efficiency. If they are not activated, resume stirring, and check more frequently until 90%activation is achieved. After the eggs settle at the bottom of the beaker, check the activation efficiency.Well-activated eggs have approximately 25%of the animal pole and 75%of the vegetal pole. Next, wash the eggs twice with a total volume of 50 milliliters of extract buffer supplemented with protease inhibitors. Then, carefully transfer the eggs to a 0.4-milliliter snap-cap microtube.Centrifuge the microtube for 60 seconds at 200 g and then at 600 g for 30 seconds to pack the eggs. Then, use a glass Pasteur pipette to remove the excess buffer on top of the eggs after each step. After this, crush the eggs at 15, 000 g for 10 minutes at four degrees Celsius.Use a razor blade to cut the tubes to separate the three layers of crushed eggs and keep the crude cytoplasm in the middle layer. Then, transfer the crude cytoplasm to clean ultracentrifuge tubes. Supplement the cytoplasm with protease inhibitors and cytochalasin B at 10 micrograms per milliliter each.Centrifuge the crude cytoplasm at 15, 000 g and four degrees Celsius for five minutes. Then, use a razor blade to cut the tube and keep the cytoplasm in the middle layer. Place the freshly prepared extracts on ice.First, add securin-mCherry mRNA to the prepared extracts. Next, add 200 microliters of 2%PFPE-PEG fluorosurfactant to the extract mRNA mixture. Use a vortex mixer to generate droplets, adjusting the vortex speed and duration as needed.Then, visually inspect the droplets to ensure they are uniform in size. Using a 200-microliter pipette, transfer the droplets floating on top and an equal volume of surfactant to a PCR tube. Dip a glass tube into the droplet layer, and wait until the droplets fill approximately 75%of the tube.Then, push the tube into the surfactant layer, and fill the tube completely. Next, fill a glass-bottomed dish with mineral oil. Then, use fine tweezers to immerse the droplet-filled tube in the oil.Use a glass pipette to remove any visible debris or leaked droplets. To avoid movement and leakage of droplets, the glass tube filled with droplets should be immersed underneath mineral oil carefully. We can either push the tube on both ends with balance or add a few drops of mineral oil on the ends of the tube simultaneously first.After this, use an epifluorescence microscope equipped with a motorized xy stage to image the droplets. Record time-lapse videos in the bright field and multiple fluorescence channels every six to nine minutes until the droplets lose their activity. Using this protocol, mitotic oscillation in both simple, nuclear-free cells and complicated cells with nuclei were produced.The nuclei-free droplets generated mitotic oscillation up to 30 undamped cycles over 92 hours. The ability of the mitotic oscillator to drive downstream mitotic events was also examined. The self-sustained mitotic oscillator drove the changes of nuclear morphology observed through the autonomous alteration of distinct cell-cycle phases.While attempting this procedure, it's important to remember to mind the time when making the extract, as it is time-sensitive, and always keep the eggs and extract on ice. After its development, this technique paved the way for researchers in the field of system biology to explore fundamental principles of complex clock properties, like tunability and stochasticity in Xenopus laevis.

reconstitution-cell-cycle-oscillations-microemulsions-cell-free

Research

JoVE Journal

Biochemistry

8.1K vues.  University of Michigan, Ann Arbor. Cette méthode peut aider à répondre aux questions clés dans le domaine de la biologie du système, telles que le mécanisme de régulation derrière le réseau mitotique et les propriétés dynamiques et les fonctions de l’horloge cycle cellulaire. Le principal avantage de cette technique est qu’elle générera une grande quantité de gouttelettes avec plusieurs cycles cellulaires dans chacun d’eux, nous fournissant un cadre à haut débit pour la manipulation quantitative et l’an...