Protocol for ‘A robust and tunable mitotic oscillator in artificial cells’ (eLife, 2018)

It is suggested to refer to our publication of ‘Reconstitution of cell-cycle oscillations in microemulsions of cell-free Xenopus egg extracts’ in JoVE (Journal of Visualized Experiments) for more detailed protocol instructions. Cite: Guan, Y., Wang, S., Jin, M., Xu, H., Yang, Q. Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts. J. Vis. Exp. (139), e58240, doi:10.3791/58240 (2018).

I. 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: pMTB2 vector backbone, securin, and mCherry through polymerase chain reaction (PCR) and gel purification.

2) Measure the concentrations of fragments using a fluorospectrometer. Combine 100ng of backbones with securin and mCherry inserts at a 1:1:1 molecular ratio and add deionized water to adjust the total volume of reaction to 5μL.

3) Prepare 6mL of 5x isothermal assembly reaction buffer with 3mL 1M Tris-HCl (pH 7.5), 300μL 1M MgCl₂, 600μL 10mM dNTP, 300μL 1M DTT, 1.5g of PEG-8000, and 20mg NAD.

4) Add the combined fragments to 15μL 1x isothermal assembly reaction buffer aliquot. Incubate the reaction in a PCR reaction tube at 50 °C for 15 minutes.

5) Make a 0.8% agarose gel and run with a voltage of 10V/cm to verify the annealing efficiency of these fragments.

6) Purify the constructed plasmid and elute using 15μL RNase-free water. Transform 1μL purified plasmid DNA into 50μL DH5α competent E. coli cells for future use.

7) Extract and purify the plasmid DNA of securin-mCherry from the DH5α competent E. coli cells.

8) Transcribe the linearized securin-mCherry plasmid DNA into mRNA and purify the mRNA. Use a spectrophotometer to verify that the concentration of mRNA is more than 100ng/μL and the ratio of absorbance at 260nm and 280nm is about 2.0.

2. Preparation of demembranated Xenopus laevis sperm DNA

NOTE: Adapted from Murray 1991.

1) Euthanize and dissect testes from four adult male Xenopus laevis.

2) Place testes from four frogs in the same 100 mL Petri dish. Rinse the testes three times in 50 mL cold 1× MMR solution (0.1 M NaCl, 2 mM KCl, 1 mM of MgCl₂, 2 mM CaCl₂, 5 mM HEPES, 0.1 mM EDTA).

3) Remove excess blood and fat from the testes and then wash twice in cold nuclear preparation buffer (NPB) (250 mM sucrose, 15 mM HEPES, pH 7.4, 1 mM EDTA, pH 8.0, 0.5 mM spermidine trihydrochloride, 0.2 mM spermine tetrahydrochloride).

4) Remove the nuclear preparation buffer (NPB) and cut testes into pieces shorter than 0.2 cm using sharp dissecting scissors. Add 8 mL of cold NPB to the testes for further breakdown.

5) Use nylon mesh fabrics with a pore size of 70 μm to filter the testes and collect sperm sample into a 15 mL conical tube. Spin down the collected sperms at 1,730× g for 10 minutes at 4 °C and remove supernatant.

6) Supply the sperm cells with 1 mL NPB and 50μL 10 mg/mL lysolecithin and incubate at room temperature for 5 min to demembranate sperm cells.

7) Wash the demembranated sperm DNA with 10mL cold NPB with 3% BSA solution three times.

8) Measure the density of sperm DNA with a hemocytometer.

9) Freeze the sperm DNA in 5μL aliquots in liquid nitrogen and store at -80 °C. The optimal concentration of sperm DNA is approximately 105 nuclei/μL.

3. Protein purification of GFP-Nuclear Localization Signal (GFP-NLS)

1) Transform the GST-tagged GFP-NLS plasmid into BL21 (DE3) competent E. coli cells.

2) Incubate the cells without antibiotics at 37 °C for 1 h and then spread on a LB agarose plate with 100μg/mL ampicillin.

3) Incubate the plate at 37 °C for 14 to 18 hours to grow the cells into single colonies. Pick and inoculate single colonies into 4 mL LB media with 100μg/mL ampicillin. Shake the cells at 37 °C for 12 hours.

4) Prepare the autoclaved YT media (16g Bacto-tryptone, 10g Bacto-yeast extract, 5g NaCl) and heat the YT media to 37 °C.

5) Inoculate 1mL of the overnight incubated cells in LB media into 250 mL preheated YT media with 100μg/mL ampicillin and shake for 2 hours to increase cell density.

6) Measure cell optical density (OD) every 30 minutes, using a spectrophotometer at a wavelength of 600 nm. Add 0.1mM IPTG into cells to induce GFP-NLS protein expression once the OD value reaches 0.1.

7) Shake the cells at 18 °C overnight to reduce cell growth rate and allow better protein expression and synthesis.

8) Spin down the cells at 34,524× g at 4 °C for 5 minutes and remove supernatant.

9) Resuspend the cell pellets in 40mL PBS buffer (supplied with 800μL 50mg/mL lysozyme, 100μL 0.2M PMSF, 13.2μL 10 mg/mL leupeptin, pepstatin, and chymostatin, and 8μL 0.5M EDTA) and incubate at 4 °C for 20 minutes.

10.) Break down cells using a sonicator at 20 kHz for 4× 30 s, with 30 s intervals between each sonication, to release expressed GFP-NLS protein.

11) Spin at 20,000× g for 35 minutes to remove the broken cells.

12) Use affinity chromatography to extract and purify GFP-NLS protein.

13) Wash the 1.5mL bead slurry three times with 15mL PBS buffer and incubate 250mL lysate with beads for 4 hours at 4 °C with inversion.

14) Spin down the beads at 2000× g for 5 min and add 10mL wash solution (25mM Tris base, pH 7.5, 500mM NaCl, and 5mM DTT) to beads. Incubate beads in 700μL of elution buffer (50mM Tris-HCl, pH 8.8, 20mM reduced glutathione, and 5mM DTT) with rotation

at 4 °C. Collect an aliquot of the elution with a volume of 50μL.

15) Measure the concentration of collected protein by running a Coomassie blue gel.

16) Elute the proteins using PD-10 columns with 3mL storage buffer (20mM HEPES, 150mM KCl, 10% glycerol, pH 7.7) through buffer exchange.

II. Preparation of Cycling Xenopus Extracts

NOTE: Adapted from Murray 1991.

1. Solution preparation

1) 0.2× Marc’s modified Ringer’s solution (MMR): 20mM NaCl, 400µM KCl, 200µM MgSO4, 400µM CaCl2, 1mM HEPES, 20µM EDTA, prepared fresh from 20× stock solution.

2) Calcium ionophore A23187: 0.1mg/mL in 0.2× MMR, prepared fresh from 10mg/mL stock solution.

3) Extract buffer: 100mM KCl, 100µM CaCl2, 1mM MgCl2, 10mM HEPES, 50mM sucrose, prepared fresh by combining 20× extract buffer (2M KCl, 2mM CaCl2, 20mM MgCl2), aliquoted 1.5M sucrose, and aliquoted 1M HEPES (pH adjusted to 8.0), then diluted with water to final concentration, pH adjusted to 7.8.

4) Cysteine solution: 2g/mL cysteine, 100mM KCl, 100µM CaCl2, 1mM MgCl2, prepared fresh by combining cysteine and 20× extract buffer stock solution, then diluted with water to final concentration, pH adjusted to 7.8.

5) Protease inhibitor solution (PI3): 10µg/mL each of leupeptin, pepstatin, and chymostatin in extract buffer, prepared fresh from diluting 10mg/mL leupeptin, pepstatin, and chymostatin stock solution (dissolved in DMSO) in extract buffer.

6) Cytochalasin B & Protease inhibitor solution: 100µg/mL of Cytochalasin B, 10µg/mL each of leupeptin, pepstatin, and chymostatin in extract buffer, prepared fresh from diluting 10mg/mL cytochalasin B, leupeptin, pepstatin, and chymostatin stock solution (dissolved in DMSO) in extract buffer.

7) Pregnant mare serum gonadotropin (PMSG): 1000U/mL in water, aliquoted and stored at -80℃.

8) Human chorionic gonadotropin (HCG): 1000U/mL in water, aliquoted and stored at -80℃.

2. Extract preparation

1) Inject three mature female Xenopus laevis with 66 IU PMSG 10 days before harvesting eggs.

2) Inject these Xenopus frogs with 500 IU of HCG to induce egg laying 18 hours before the preparation of cycling extracts.

3) Squeeze the eggs of each female frog into 100 mm Petri dishes containing 10 mL of 0.2x MMR buffer and inspect under a stereo microscope. Discard eggs laid overnight. Based on experiments (data not shown), extracts prepared from the freshly squeezed eggs generate longer lasting activities than extracts prepared from laid eggs from the same female frog.

4) Choose the batch of eggs from one female frog that appears homogeneous and with distinct boundaries between animal and vegetal poles and transfer the eggs into a 600 mL beaker. Discard the batches of eggs that have unclear boundaries between animal and vegetal poles or have irregular white spots on top of animal poles.

5) Pour out excess 0.2x MMR buffer and gently add 250 mL cysteine solution to eggs.

6) Shake the beaker vigorously by hand to remove the jelly coats of eggs with 3 doses of cysteine solution, a total volume of 800 mL for 3 minutes or until eggs physically touch each other and settle at the bottom of the beaker.

7) Wash eggs with 4 doses of of 0.2x MMR solution, a total volume of 1L.

8) Pick and discard eggs that turn white using a glass transfer pipette.

9) Pour out the MMR buffer and supply eggs with 200 mL calcium ionophore solution for activation.

10) Use the wide opening side of a glass pipette to stir the eggs gently until the eggs are activated (Usually after 1~2 min). Well-activated eggs have approximately 25% of the animal pole and 75% of the vegetal pole. Check the activation efficiency after eggs settle down at the bottom of the beaker, then remove calcium ionophore solution.

11) Wash the activated eggs twice with PI3, a total volume of 50 mL .

12) Carefully transfer eggs to a 0.4 mL snap-cap microtube (prefilled with cytochalasin B & Protease inhibitor solution) and then pack eggs through centrifugation at 200×g for 60 seconds, and then 600×g for 30 seconds in a tabletop centrifuge.

13) Remove extra buffer on the top of eggs using a glass Pasteur pipette to reduce the dilution of extracts after both centrifugations.

14) Crush eggs with high speed centrifugation at 15,000× g for 10 minutes at 4 °C. The content of the eggs should be separated into 3 layers: the upper yellow lipid layer, the middle transparent cytoplasm layer, and the lower dark yolk layer.

15) Cut the tubes with a razor blade to separate three layers of crushed eggs and keep the middle crude cytoplasm layer.

16) Add PI3 (10mg/mL in DMSO) and cytochalasin B (10mg/mL in DMSO) to the crude extract so the final concentration of each is 10µg/mL. Mix well then transfer to new 0.4 mL snap-cap microtubes.

17) Centrifuge the crude cytoplasm again at 15,000 x g at 4° C for 5 minutes to further purify cytoplasm by removing excess lipid and yolk.

18) Cut away the top and bottom layer with a razer blade again and keep the middle extract layer.

19) Keep freshly prepared extracts on ice.

III. Droplet Generation

1. 2D glass chamber preparation

NOTE: The rectangle glass tubes serve as chambers that confine the droplets in a single 2-dimensional plane, allowing for easier observation and data collection.

1) Cut rectangle glass tubes (inner dimension 2mm × 100μm) into 4 mm long pieces. Use the sharp edge of a whetstone, mark the desired boundaries on the outer surface of the glass tube with light etchings, then gently apply pressure on both sides of the etchings to break off pieces of glass tubes.

2) Pre-heat a dry bath incubator to 95°C. Take out the heating block and place it into a polycarbonate vacuum desiccator.

3) Place a 1.5mL microcentrifuge tube containing 30μL trichloro(1H,1H,2H,2H-perfluorooctyl)silane in the heating block. Lay the cut glass tubes inside the vacuum desiccator at approximately 5cm to the heating block.

4) Connect the desiccator to a vacuum pump. Turn on the pump for 1 minute to reach the desired vacuum level, then close the 3-way valve to seal the desiccator and let the trichloro(1H,1H,2H,2H-perfluorooctyl)silane vapor coat the inner wall of the glass tubes. This will create a hydrophobic environment for stabilizing the droplets.

5) Leave the glass tubes inside the desiccator for overnight coating. The tubes will be ready for use the next day.

2. Droplet generation and imaging

1) Supply extracts prepared in step II with 10ng/μL securin-mCherry mRNA for the simple cytoplasmic-only cell cycle experiments. Alternatively, supply extracts with demembranated sperm chromatin (250 per μL of extract), 10μM GFP-NLS protein, and 10ng/μL

securin-mCherry mRNA to create droplets exhibiting periodic nuclei morphology changes.

2) In a 1.5mL microcentrifuge tube, mix 20μL of extract supplied with recombinant protein or mRNA with 200μL of HFE7500 fluorinated oil with 2% perfluoropolyether-polyethylene glycol (PFPE-PEG) fluorosurfactant.

3) Generate droplets using a vortex mixer. The range of droplet sizes can be modulated by vortex speed and duration. To create droplets with radii ranging from 50 to 200μm, set the vortex speed at 56× g (3,200 rpm with a radius of 4.9 mm) and gently press the microcentrifuge tube containing the extract and surfactant mixture on the mixer for 3.5 seconds. Visually inspect if the droplets are uniform in size.

4) Use a 20μL pipette to transfer the droplets floating on top and an equal volume of PFPE-PEG surfactant into a PCR tube. Dip the glass tube prepared from step 1 into the droplet layer in 1.5mL microcentrifuge tube and wait until droplets have filled approximately 75% of the tube, then push the tube down into the surfactant layer until the tube is completely filled. This will lower the droplet density and allow the droplets to form one single layer inside the chamber without overlapping or shape distortion caused by overcrowding.

5) Fill a glass bottom dish (diameter 40 mm) with mineral oil. Immerse the droplet-filled tubes in oil by gently pushing them down using a fine tweezer.

6) After loading all samples, use a glass pipette to remove any visible debris or leaked droplets. Add more mineral oil if the tubes are not or will not be completely immersed in the imaging process.

7) Conduct imaging of droplets on an epifluorescence microscope equipped with a motorized x-y stage. Use a 4× air objective for all imaging. For fluorescence imaging, use a 130W mercury vapor short arc lamp as fluorescence light source for illumination, and a GFP filter set (excitation 482/35 nm and emission 514/LP nm) and an mCherry filter set (excitation 562/40 nm and

emission 641/75 nm) for imaging GFP-NLS and securin-mCherry signals.

8) Record time-lapse videos in the bright field and multiple fluorescence channels every 6 to 9 minutes until the droplets lose their activity.

IV. Image Processing and Data Analysis

1. Segmentation and tracking of droplets

1) Import recorded data in the format of ".tif" or ".oif" into image analysis software.

2) Segment single droplets using the bright field channel. The bright field image will show droplets as bright circles with dark boundaries. Apply thresholding to the image. Add a tracking surface to each droplet by tuning the threshold so that each surface covers the whole area of the corresponding droplet.

3) Automatically track the segments between adjacent frames using the autoregressive motion algorithm. Tune the acceptable maximum traveling distance of a droplet between two adjacent frames to be smaller than the radius of most droplets to accurately track small droplets.

4) Visually check all tracks across the entire video to detect incorrect segmentations that marks the empty space between droplets as actual droplets. Manually delete incorrect tracks.

5) Segment and track background area without any droplets to obtain the background fluorescence intensity. To improve signal to noise, subtract background intensity from the fluorescence intensity of droplets at each time frame.

6) Export measured parameters for each track over time, including the mean and standard deviation of the fluorescence intensity and the droplet area into ".csv" files.

2. Data analysis of droplet size and oscillation period

1) Load the ".csv" files into R to create plots of fluorescence intensity over time for each droplet. Record droplet ID into a ".csv" file.

2) Import the ".csv" files exported from the analysis software and the ".csv" file with a list of droplet ID into Matlab and save as ".mat" files for further data analysis.

3) Calculate the volume of the droplets based on the exported droplet area information after segmentation and the height of glass chamber. Use the volume to calculate the radius of droplet as a sphere.

4) Extract the time points of peaks and troughs using peak/trough detection algorithm. Perform manual corrections on the position of peaks and troughs to ensure they are accurately detected.

5) To calculate the cell-cycle period, calculate the interval time between two adjacent peaks. Take the average of all intervals for each droplet as its cell-cycle period.