With a simple mix of ingredients and functionalized surfaces, this protocol recreates a vital function of cells, movement powered by actin assembly. The biochemical and biophysical mechanisms of force production can be assessed by varying the ingredient mix and the properties of the functionalized surface. The main advantage of this protocol is that all the ingredients can be purchased, so expertise in protein purification is not necessary.
This allows non-specialists to use this system as a tool in their research. This protocol provides an undergraduate-level teaching module, giving students hands-on experience manipulating proteins and observing a self-prepared biological system under the microscope. They can control and vary system parameters.
Physical analysis, such as a Langmuir equation, can be run on trajectories. Begin by resuspending lyophilized proteins. Gather the solid at the bottom of the tube by pulse centrifuging protein powders at four degrees Celsius.
Then add water as per the manufacturer's instructions and incubate on ice for at least 15 minutes. Mix gently by pipetting. Keep it on the ice for another 15 minutes and mix again.
Collect the solution at the bottom of the tube by centrifugation. Remix, and aliquot on ice. To depolymerize actin oligomers formed during lyophilization and freezing, dilute an aliquot of resuspended actin eight-fold in G-buffer spiked with additional ATP, DTT, and 10%labeled actin.
Allow it to depolymerize on ice with occasional mixing for at least a few days to a week before measuring protein concentration. To measure protein concentrations of resuspended proteins, first prepare a BSA dilution series. Start by placing two milliliter and 1.5 milliliter tubes in rack as described in manuscript.
Fill a 15 milliliter conical tube to the top with Bradford Reagent and place it on ice. Take 200 microliters of Bradford Reagent and eject it back into the conical tube to wet the pipette tip. As the solution is viscous, pipette slowly to allow the solution to enter and leave the tip completely without making bubbles.
Then using the pre-wet tip, pipette 200 microliters of Bradford Reagent into each of the 1.5 milliliter tubes in the rack. And return the remaining content of the 15 milliliter conical tube to the bottle. Measure out water into the two milliliter tubes of rows one, three, and five.
Prepare the BSA dilution series by diluting the calibrated BSA stock solution as described in the manuscript. Then make serial dilutions by transferring 900 microliters of each solution into the next tube. Add 800 microliters of water to the Bradford Reagent tube for the blank and start the timer.
Mix 800 microliters of each BSA standard with 200 microliters of Bradford Reagent in the prepared tubes. Within five minutes after mixing with the Bradford Reagent or each standard in a disposable cuvette then read the absorbance at 600 nanometers. Graph the obtained absorbance values as a function of known BSA concentration and obtain a linear correlation with an R value of 0.99.
In the tubes containing 2000 microliters of water, dilute resolubilized proteins gently as described in the manuscript. Immediately add 800 microliters of each solution to the prepared Bradford Reagent and mix. Read the absorbances in the spectrophotometer within five minutes.
Calculate protein concentrations using the previously constructed standard curve. For bead coating, pre-chill the centrifuge to four degrees Celsius. Pipette 50 microliters of Xb buffer into a 1.5 milliliter microcentrifuge tube.
Thoroughly mix 4.5 micrometer diameter bead suspension by vortexing and add nine microliters of the suspension to the tube containing Xb buffer. Centrifuge the samples at 20, 000 G for 10 minutes at four degrees Celsius. To coat the beads, carefully remove the supernatant without disturbing the beads and resuspend the bead pellet in 40 microliters of two micromolar SpVCA in Xb buffer by gently pipetting.
Now agitate the beads in the dry block at 1000 RPM for 20 minutes at 18 degrees Celsius. Next, wash the coated beads by centrifuging. Removing the supernatant and resuspending the beads in 50 microliters of cold Xb with 1%BSA.
After washing the beads, resuspend the coated bead pellet in 120 microliters of cold Xb, 1%BSA. Store on ice in a refrigerator or cold room. Prepare the motility reaction mixture as described in the manuscript.
Quickly mix the reaction and start the timer. Spot the entire motility reaction mixture on a slide. Cover it with an 18 millimeter by 18 millimeter coverslip and seal the coverslip with melted VALAP using a small paintbrush.
To obtain average displacement speeds for a whole population of beads, record phase contrast or fluorescent still images over time by scanning the entire slide. Measure comet length by hand and log the values. Plot the comet length versus time.
The slope of the linear fit is the average growth speed. Select time lapse movies in phase contrast microscopy to evaluate individual bead speeds. Depending on the bead speed and the resolution required, take frames every 1 to 10 seconds.
Use the tracking tool of any image processing program to obtain bead speeds and trajectories. Mixing BSA standards with Bradford Reagent gives a solution with graded blue hues. Absorbance values are plotted versus the standard protein concentrations and the linear correlation is used to determine resuspended protein concentrations.
Mixing SPVCA-coded beads and motility medium containing capping protein leads to actin cloud formation within minutes. Cloud polarization occurs at approximately five minutes and comet production at 15 to 20 minutes. The actin comets continue to elongate for many hours but a consistent speed is not maintained.
So bead motility is evaluated within one hour. Motility mix with either capping protein or gelsolin gives comets in a similar manner. Actin clouds polarized to form comets in the first 20 minutes of reaction and comets elongate over time.
Evaluation of comet lengths measured over time is used to calculate displacement speed. In the absence of capping activity, actin clouds form around beads, but comet formation does not occur. Careful handling and pipetting of proteins is essential for success of this procedure, not only when making the motility mix, but also when measuring protein concentrations with the Bradford assay.
Comet formation and bead trajectories obtained with this protocol allow for understanding the physical mechanism of movement using soft matter and statistical physics analysis and biophysical experimentation, including force measurements and micromanipulation.
