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Bio130 Lab 1: Actin Filament Assembly Purpose: To use a simple quantitative assay to monitor actin assembly. Class data will be

pooled, providing a large data set for analysis. Actin polymerization An actin filament (F-actin) is a helical polymer of the monomeric globular protein, actin, or G-actin (Figure 1). The filaments are flexible structures with a diameter of about 7 nm. They are organized into linear bundles, two-dimensional networks, and three-dimensional gels within eukaryotic cells, although they are most highly concentrated in the cortex, the region of cytoplasm just beneath the plasma membrane. Actin filaments are dynamic structures whose assembly and disassembly are critical for cellular functions. Prokaryotes have analogous (not homologous) cytoskeletal proteins. Actin filaments perform a variety of functions, including cell movement and membrane movement at the cell surface (e.g., phagocytosis). They create cell surface microvilli, contractile bundles in the cytoplasm, sheet-like or fingerlike projections (lamellapodia or filopodia), and the contractile ring during cell division. Actin is a critical component of muscle, creating filaments across which the motor protein, myosin, moves to cause muscle contraction. A large number of actin binding proteins regulate filament assembly, disassembly, and availability to other binding proteins in order to accomplish such varied tasks. Actin Binding Proteins Actin is the most abundant protein in many eukaryotic cells. The study of actin polymerization is of interest because actin only functions in filamentous form. Thus, the control of where and when actin assembles in cells is important for determining the shape and motile properties of a cell. In muscle cells most of the actin is polymerized but in non-muscle cells about 50% of the actin remains un-polymerized. Non-muscle actin filaments also assemble and disassemble more frequently than muscle actin filaments. Actin binding proteins (ABPs) have been identified that can aid in actin polymerization or depolymerization, change the length of filaments, and cap one end of actin filaments. The Viscometry Assay Polymerization of actin from monomers to filaments can be measured conveniently by monitoring changes in the viscosity of an actin solution. A solution of actin filaments has a higher viscosity than one containing actin monomers because they are large and asymmetric. One can, therefore, measure the amount of time it takes a steel microball to fall through a solution of actin as an indication of the amount of actin polymerization in that solution. Viscosity changes of an actin solution can also be used to screen crude cellular extracts for the presence of factors that regulate the assembly state of actin filaments. We will use this viscometry assay to measure actin polymerization over time and to measure the activity of actin binding proteins such as coronin or cofilin.

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Step 1: Learning to Pipette Proper pipetting technique is critical to the success of this lab, and to lab work in almost all areas of biology. Each person should become comfortable with the pipettes using the colored water at your station. The instructor and lab assistant will come by and check your work.

1. Set the desired volume on the pipette. Do not force the dial beyond the volume printed on the top of the pipette plunger!

2. Pick up a sterile pipette tip with the pipetter – blue tips = P1000; yellow = P200 or P20. 3. Depress the plunger with your thumb to the first stopping point, NOTE: do not push

beyond this point or you will fill the tip with a larger volume than you intend to use. 4. Insert the tip into the solution you wish to move. Do so at eye level (yes, really. Think

of it as deltoid exercise). 5. Lift your thumb up slowly to avoid getting air bubbles in the tip (air bubbles will

decrease the volume you are trying to carefully measure). 6. Again at eye level, move pipette tip to new tube and dispense the liquid. This time

pushing the plunger all the way down, past the ‘squishy’ stopping point to expel the last tiny drop. Eject the pipette tip into a white waste bucket.

7. Test your pipetting skill – Weigh 500 µl of water. Try 200 µl with a P200. Is it right?

Experiment 1: Measuring actin polymerization over time 1. Set up the inclined platform that has been provided to you. Stick a small piece of clay to

the surface to act as a pipet holder and smear a small dab of modeling clay on your ceramic tray. This will be the area you use for sealing the end of your capillary tubes.

2. One student on each team should practice pipetting 60 µl of sterile water from a 500 µl

eppendorf tube into a 50 µl capillary tube in a controlled fashion. You want the water to reach past the black line on the capillary tube but you don’t want any air bubbles in the tube. Hold the capillary tube and maintaining constant pressure with the silver pipette screw, seal the free end of the tube with the modeling clay by sticking the tube into the clay and sliding it sideways.

3. Fix the capillary tube to the inclined plane with the larger piece of modeling clay so that

the top opening of the tube extends beyond the top of the plane. Be sure the angle of the capillary tube is the same as that of the inclined plane.

4. To measure the viscosity of the water, insert one steel microball into the open end of the

pipet using your fingers and a magnet to prevent the ball from falling into the solution prematurely. Controlling the ball with a magnet, slowly move the ball to the black line on the capillary tube.

5. The second person should use a stopwatch or cell phone timer to determine the number of

seconds it takes for the ball to reach the bottom of the capillary. The person holding the ball steady with the magnet should move the magnet to release the steel ball and say ‘start’ and tell the partner to ‘stop’ when the ball reaches the bottom. Practice this several times until you have your technique down. Do at least 3 replicate measurements of the viscosity of water and record your data. Remove the steel ball using the magnet and re-use it for the zero time point.

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6. The second person should be responsible for labeling and preparing the set of 5 reactions

below in 500 µl eppendorf tubes – one at a time. Also label a spot where the sealed capillary tubes will incubate before measuring viscosity – use your modeling clay to fashion a spot where they can hang – they tend to leak when the capillary tubes sit inside a plastic tube. This person should also be responsible for creating and filling in a data sheet and timing.

7. Set up the 70 µl reaction mixture starting with the 0 time point. DO NOT proceed to set

up any other reactions until you are comfortable with following the procedure using the 0 time point. Mix together the components for the 0 time point in a 500 µl eppendorf tube according to the following chart:

Overview of all reaction mixtures (add reactants from left to right):

Reaction # 10X initiator dH2O G-actin (2 mg/mL) Incubation Time (minutes)

1 -- 50 µL 20 µL 0 2 7 µL 43 µL 20 µL 45 3 7 µL 43 µL 20 µL 25 4 7 µL 43 µL 20 µL 15 5 7 µL 43 µL 20 µL 2

8. Immediately after mixing the reaction by flicking the tube with your finger, tap the tube

on the bench top so that all of the drops are at the bottom. Have your partner carefully draw the liquid up into a capillary pipet using the silver screw pipetter. Draw liquid up past the black line, but not into the pipetter. Seal the end of the capillary with modeling clay as before. Put the capillary on the inclined plane. Add a steel microball and hold it at the black line until your partner is ready to starting timing.

10. Move the magnet away from the capillary pipet to release the ball. At the same time that the ball is released, start the stopwatch. It is very important that you are timing the ball falling the SAME DISTANCE for each of your reactions. Once you have measured this viscosity, draw the ball back up to the black starting line and repeat the measurement. Collect 3 such measurements for each reaction.

Note: For the 0 time point, you substituted 10 µl distilled water for the initiator since it would

otherwise be impossible to measure a true "0" time point. 11. Next set up the 45, 25, 15 and 2 min incubations, in this order. All 4 can be set up before

measuring as long as you set up the 2-minute incubation last! Draw up each reaction in the pipet, seal the bottom of the pipet, start timing the incubation, and place the pipet in a safe place until the reaction has gone for the indicated time and is ready for the falling ball. For the 2-minute incubation, seal the pipette, starting timing 2 minutes, and attach the pipette directly to the inclined plane. Add the steel microball and hold it at the black line with the magnet until the 2 minutes are up. Then drop the ball and start the stopwatch. Do not let the ball drop through the solution prematurely. If you do, start timing 2 minutes over again.

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12. After measuring the falling ball rate for the each time point, bring the ball back to the top

of the solution immediately with your magnet and measure the rate again right away. Is the rate different? Why or why not? Measure the falling ball rate a third time as well.

13. Move your data to the Excel spreadsheet on the lab computer. Indicate with a * any

measurement that required you to jostle the ball with a magnet to get it going again. Experiment 2: Measuring the effect of ABPs on actin polymerization For this experiment, the variable will be an unknown actin binding protein (ABP). 1. Mix together the components for each reaction in a 500 µL tube according to the following chart, perhaps 3-5 minutes apart (add reactants from left to right):

Reaction # 10X

initiator dH2O ABP fraction

(0.04 mg/ml) PBS w/ glycerol

G-actin (2 mg/mL)

Incubation Time (min)

1 7 µL 33 µL 10 µL -- 20 µL 45 2 7 µL 23 µL 20 µL -- 20 µL 45 3 7 µL 13 µL 30 µL -- 20 µL 45 4 7 µL 13 µL -- 30 µL 20 µL 45

2. After mixing the reaction mixture by flicking with your finger, tap the tube on the bench top so that all of the drops are at the bottom and draw the liquid up into a capillary pipet using the mouth tubing and seal the end of the capillary tube as before. Set your filled capillary tubes in labeled 1.5 ml eppendorf tubes and be sure to note when you started each incubation. 3. Measure the relative viscosity of the solution using a steel microball as before. Again, it is very important that you are timing the ball falling the SAME DISTANCE for each of your incubations. Again, take three measurements for each sample. 4. Record your data in the Excel spreadsheet on the lab computer. Indicate with a * any measurement that required you to jostle the ball with a magnet to get it going again. Notes: 1. Pipet solutions carefully and precisely to avoid introducing a new variable. 2. Always add components in the correct order (left to right from each table above). 3. All incubations should take place in the capillary tube. 4. When you draw the reaction up into the capillary tube, stop when the liquid is flush with the

bottom of the microcapillary pipet so you do not end up with an air bubble at the bottom. 5. Capillary tubes will break if pressed too hard into the platform. Take Care. 6. Keep capillary tubes parallel with the slant of your platform so the angle is constant. 7. Be prepared for the ball to get stuck and nudge it gently with the magnet. 8. Save the microcapillary pipettes containing the steel microballs. The steel microballs are very expensive and will be washed and re-used (yes, really!).