Actin Myosin Motility Assay

(& Actin Filament Length Dependence on ATP)

Katie Munns and Thomas Vennemann

Purpose:

The goal of this project was to determine the maximum velocity of actin filament movement due to myosin motor proteins in the presence of adenosine triphosphate (ATP). Data on the initial goal regarding the motility assay was not able to be collected due to time constraints resulting from first needing to find the optimal environment at which to run such an experiment. Therefore, the aim was changed to determining the optimal environment for actin polymerization. Just as our bodies perform best in certain conditions, actin, which are just one of the pieces of our muscular anatomy, will also polymerize best in certain conditions. The conditions are solely based on the type of buffer used and the concentration of ATP present. We will go over some of basic theory behind this experiment, as well as the buffers utilized and the ATP concentrations used for the polymerization.

Theory:

Any enzymatic reaction in biology follows the Michaelis-Menten theory. In the polymerization of actin, there are three main components; actin monomers (G-actin), actin filaments (F-actin), and ATP. To form the filaments, ATP binds to a specific binding site on the actin and is hydrolyzed to ADP and a phosphate ion. When the non-binding side of an actin monomer (or filament) comes by the ATP bound actin, the phosphate ion releases and a bond between the actin is formed. This process happens constantly as long as binding sites are available and ATP is present in the system.

In theory, as the ATP concentration is increased, the polymerized actin lengths will also increase until some saturation limit is reached. This means all actin binding sites are filled and there is now an excess of ATP floating in the system. In this project it is key to understand what this saturation limit is, and we'll explain why later on.

If this short explanation doesn't make a lot of sense, this wikipedia page explains the basic theory very well and gives images: http://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics

Experimental Procedure:

There are two overarching procedures in this experiment. The first is the actin polymerization and the second is the actual Motility Assay. We will go over some of the things we've attempted with both of these, and some suggestions on what to do in the future.

Actin Polymerization

To polymerize the purified G-actin, we do the following steps:

• 1. 1. Re-suspend the actin to 1 mg/mL (starting with 5 µL of 4 mg/ml)

     1. 1. a. 0.2mM ATP (0.522 µL)

        2. b. 1.0mM DTT (2.4 µL)

        3. c. General actin buffer (177.08 µL)

        4. d. Mix a, b, and c.

        5. e. Take 1.5 µL of d and add to 0.5 µL of actin

        6. f. Mix well and leave on ice for 1 hr.

  2. 2. Polymerize actin with 1/10^th the volume poly-buffer

• • • 1. a. Take XXX µL ATP and add YYY µL (One of the buffers made) to obtain 0.01 M (Buffer Name)*

      2. b. Add ZZZ of (Buffer Name) to result from step 1*

      3. c. Add about 0.2 µL phalloidin

      4. d. Leave at room temp for 1 hour

This is just one procedure we came up with that was fairly similar to most papers. Step 1 can be omitted since this solution is basically what is already present in the G-actin solution bought online. The only purpose of it is to dilute the actin to 1 mg/ml and to prevent clumping of the actin (which is done by the addition of DTT which breaks apart the disulfide bonds). Step 2 is the most important step to get correct. The amount of ATP we would introduce here is crucial to the polymerization of actin. This is why the values are XXX, YYY, and ZZZ. The only thing that's important here is the molar ratio of ATP to Actin. The point of this part of the experiment is to determine the "Optimal Environment", and varying these parameters is how the problem is solved.

The buffers we used are as follows:

• 1. 10x Poly-buffer

     1. 1. 500 mM KCl

        2. 20 mM MgCl₂

        3. 10 mM ATP (Added in day of usage)

  2. 10x AB Buffer, pH 7.4

• • • 1. 25 mM Imidazole Hydrochloride

      2. 25 mM KCl

      3. 25 mM MgCl₂

      4. 25 mM EGTA (ethylene glycol tetraccetic acid)

      5. 25 mM DTT (dithiothreitol) (Added day of use)

• 3. 4x F-Actin Buffer, pH 7.0

• • • 1. 300 mM KCl

      2. 10 mM MgCl₂

      3. 40 mM HEPES

• 4. General Actin Buffer (Only used in initial step, not tested for polymerization)

• • • 1. 5 mM Tris-HCl (pH 8.0)

      2. 0.2 mM CaCl₂

• 5. ABSA Buffer

• • • 1. 10% BSA

      2. 10% AB Buffer

      3. 80% DI water

The composition of these salts should remain constant in the experiment, however, it is up to the discretion of the experimenter if they want to alter concentrations of the salts and buffers used in each specific buffer. The addition of salts is key for polymerization and having a stable pH of between 7.0 and 8.0 is also essential, finding out what pH works best is also one of the optimization issues.

Motility Assay

For the motility assay, we use the following procedure

• 1. 1. Dilute polymerized actin filaments (XX) fold in 1x Buffer

     1. 1. a. Add 100 µL of buffer to dilute filaments to the resulting volume from step 2

  2. 2. Prepare the myosin aliquot from myosin stock

• • • 1. a. 1 µL myosin+99 µL DI water (or a buffer) (a procedure says to use AB buffer)

• 3. 3. Fabricate slides

• • 1. a. Clean slides with soap +water, ethanol, DI-water rinse, nitrogen gas to dry, and then spread nitrocellulose and allow to dry

       1. 1. nitrocellulose is prepared by making a 1% solution of it diluted in acetone

    2. b. Put down double stick tape channel

    3. c. Put down coverslip (slide fabrication shown below)

• 1. 4. Pipette 40 µL myosin into channel

     1. 1. a. Wait 10 min.

  2. 5. Pipette 100 µL ABSA buffer into channel to rinse and block sites myosin didn't bind to

  3. 6. Prepare Motility Solution

     1. 2. 1. Mix diluted actin with ATP.

           2. ***This is where the molar ratio of ATP over actin is important and is varied.

  4. 7. Pipette 40 µL motility solution

• • • • 1. Periodically to replenish depleted ATP if motion is seen with same concentration used previously.

Results:

When testing which buffer would supply the best environment, three were chosen and compared against one another. They were the: (I) Polymerization Buffer, (II) AB Buffer, and (III) F-actin Buffer. Shown below is a picture of the monomeric actin seen in the F-actin buffer under a single molecule resolution microscope. This picture was taken after the polymerization step, and one can see that no polymerization took place.

Pictured above: F-actin monomers

The best buffer ended up being the AB Buffer, which we then used in some preliminary testing of the dependence of actin filament length on ATP concentration.

Future work would entail finding the limiting length, or saturation point of the ATP.

Pictured: Actin in 2μM ATP

in 200μM ATP

in 1mM ATP

One can observe the increase in number of observable actin filaments as well as an increase in filament length with increasing [ATP]. The average lengths were: (2.0±0.2)μm in 1mM ATP, (1.46±0.04)μm in 200μM ATP with nitrocellulose, (1.69±0.07)μm in 200μM ATP without nitrocellulose, and (0.65±0.05)μm in 2μM ATP.

Concluding Remarks:

In this project, the motility and polymerization shouldn't be done on the same day. The initial goal should be to optimize polymerization and when that is done, then use only those optimum conditions to polymerize. That, now perfected actin polymerization, can be used in the motility assay. In the actual motility assay, the myosin heads use ATP to push the actin along, so the concentration of ATP used in this part will abide by the same theory as discussed in the actin polymerization. Testing a large array of ATP concentrations here will allow the experimenter to see a wide array of actin filament speeds and when data is taken, the array of velocities measured should allow for a Michaelis-Menten curve to be generated.

Acknowledgements:

We would like to extend a thank you to our professors and peers in MXP II for all their support, with a special thanks to our advisor Professor Elias Puchner for all his guidance and for setting up the single molecule resolution microscope on such short notice.

References (which could prove useful for future work):

[1] (2013, July 23). Motillity Assay Enzyme Kinetics and Molecular Motors. Lab Course G3. Conducted from LMU Munich Physics Department, Chair for Applied Physics Gaub Lab.

[2] Davies, R. (1963). A Molecular Theory of Muscle Contraction : Calcium-Dependent Contractions with Hydrogen Bond Formation Plus ATP-Dependent Extensions of Part of the Myosin-Actin Cross-Bridges. Nature, 199, 1068-1074.

[3] Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 18.2, The Dynamics of Actin Assembly.

[4] Kron, S., & Spudich, J. (1986). Fluorescent Actin Filaments Move on Myosin Fixed to a Glass Surface. Proceedings of the National Academy of Sciences, 83, 6272-6276.

[5] Sellers, J. (2006). In vitro Motility Assays with Actin. In J. Celis (Ed.), Cell Biology: A Laboratory Handbook (3rd ed., Vol. 2, pp. 387-392). Amsterdam: Elsevier Academic.

[6] Coluccio, L., & Tilney, L. (1984). Phalloidin enhances actin assembly by preventing monomer dissociation. The Journal of Cell Biology, 529-535.

[7] Burlacu, S., Janmey, P., & Borejdo, J. (1992). Distribution of actin filament lengths measured by fluorescence microscopy. American Journal of Physiology-Cell Physiology, 262(3).