User:Andy Maloney/Water isotope effects on kinesin and microtubules
This page describes the experiment where I investigated gliding speed variations of microtubules using different water isotopes.
This is the third chapter in my completely open notebook science dissertation. If you would like to post questions, comments, or concerns, please join the wiki and post comments to the talk page. If you do not want to join the wiki and would still like to comment, feel free to email me.
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Methods and materials
Most of this section is identical to previous chapters. I will only describe the differences done with this assay in this section below while linking to the relevant components from other chapters. I am doing this in case someone reaches this page through a search engine and not by traveling through the natural progression of the thesis that this chapter is attached to.
- My microscope setup and the programs I used to analyze the data can be found in Chapter 1. A description to microtubule length measurements can be found in Chapter 2's Results section, specifically in Figure 15 of that chapter.
- Temperature stabilization is still crucial for stable speed measurement and a description can be found in Chapter 2's Methods and materials section.
- The basic flow cell construction can be found in Chapter 1's section 3. I did modify the basic flow cell somewhat for a specific assay done in this experiment. I will describe in detail what I did to construct this new flow cell below.
- Basic buffer solutions can be found in Chapter 2's section 3 as well. For this experiment, I changed the solvent that the kinesin and microtubule system was in. I will describe in detail the differences I made to the basic PEM solutions below.
- Microtubule polymerization, as well as the experimental setup, can be found in Chapter 1's Experiment section. There are a lot of things one can do with polymerization of microtubules and I will discuss some of these things in the Future work section of this chapter.
The differences for these experiments as compared to others I've done are subtle, however, they make a big difference to the assay. The measurable differences in how the assay responds to different environments is not too surprising considering how sensitive it is to ionic strength and pHCite Bohm.
New flow cells
The construction of the new flow cells was very similar to the old construction. The only difference was that I intended for the new flow cells to not be permanently sealed. This required finding a material that would "seal" the flow cell in order to prevent evaporation and at the same time allow me to observe it on the microscope, remove it from the microscope, and flow in new material to be observed again. Surprisingly enough, plain old kitchen plastic wrap (cellophane) worked the best at this task.
|Step 1: I created a template for fast and reproducible flow cell creation. Using my template, I centered a slide (VWR 48300-025) in the vertical rectangle.|
|Step 2: I added strips of permanent double stick tape (Scotch) to the slide by using the two most center lines as a guide. This created a channel that is approximately 10 µL in volume. The width of the two center lines was 5 mm.|
|Step 3: I trimed the excess tape by using the outer lines of the center box as a guide. I did not trim the tape such that the only tape left on the slide was in the center box. I only trimed the tape that hung over the slide. It turns out that keeping this extra tape is useful when making resealable flow cells. After trimming the tape I placed a cover slip (VWR 48366-045) over the center box. The center box is used as a guide to ensure that the cover slip is centered on the slide.|
|Step 4: I ensured proper adhesion of the cover slip to the tape by pressing it to the slide. Proper adhesion was done when the light that is scattered from the slide + tape + slip combo differs from when the slip is merely placed on the tape. Compare the figure from step 3 to this step and notice the difference in how the light scatters from the center box.|
|Step 5: If the flow cell was not intended to be used as a resealable one, then it was finished with the previous step. If it was intended to be used with the cellophane sealer, then I continued on with the following steps. Two very thin pieces of double stick tape are placed at the entrances of the flow cell. They have been colored red in the image to enhance contrast. These pieces of tape are essential for proper sealing with a piece of cellophane and help prevent objective oil from seeping into the flow cell. They can be made by holding two razor blades together and cutting a thin piece of tape with them. I had to ensure that the thin pieces of tape were as close as possible to the entrances of the flow cell since I observed excess evaporation occur if they were not close.|
|Step 6: Small strips of cellophane (Glad Cling Wrap) were then placed over the flow cells and wrapped around the slide.|
I should note that I also tried static cling vinyl and pallet wrap before settling on using cellophane. The static cling vinyl was not used because it was too thick and did not properly seal the flow cell. But, it did indicate to me that the static cling was what I wanted to use as the new sealer for the flow cells. This lead me to pallet wrap which was not used as well because it contains a small amount of glue on it and I did not want that glue seeping into my flow cells. Using it did show me that I needed a thinner material to "seal" the flow cell with and ultimately lead to the use of cellophane that can be purchased at a grocery store.
There has been some debate about the use of nail polish as the sealant for flow cells due to the organic solvents that are in them. Using both the nail polish sealant method and this new cellophane seal method which has no chemicals that can leach into the flow cell showed no difference in the observed gliding motility speeds. I will discuss this in greater detail and show a figure below in the Results section.
Along with the 10x concentrated solution of PEM mixed in 18.2 MΩ-cm water, I prepared a 10x concentrated solution of PEM in D2O (Sigma 151882) using 800 mM PIPES, 10 mM EGTA, and 10 mM MgCl2. I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct "pD" of this solution using the pH meter, I had to add 0.41 to the measured value cite Covington and Paabo. This necessitated that the solution of PEM in D2O was pH-ed to 7.30 using an approximate amount of 1.25 M NaOH. I designated this solution as HPEM to indicate it was the heavy hydrogen water buffer. It was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4˚C.
A third and final buffer containing PEM was prepared with H218O (Sigma 329878). H218O, or heavy oxygen water, was prohibitively expensive to prepare a 10x concentrated solution. Instead, I opted to dilute the regular 10x PEM in the heavy oxygen water such that the final concentration of salts and buffers in this solution was the usual 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2. I designated this solution as OPEM to indicate that it was the one using the heavy oxygen water. This solution was not pH-ed and it was not filtered for fear of loosing too much material in the filter. It was aliquoted and stored at 4°C.
Experiment and data analysis
The experimental procedure and data analysis is nearly identical to the steps taken in Chapter 2. Below, I will outline the experimental procedure since it does differ from what was done in Chapter 2 and is relevant to the discussion below. However, I will only link to the data analysis section of Chapter 2 below.
Experimental flow cells for observation were prepared as follows.
- Prepared a flow cell. Either the type to be sealed with nail polish or the type to be sealed with cellophane.
- Incubated the flow cell with 1.0 mg/mL alpha casein in PEM for 10 minutes.
- During the 10 minute incubation, kinesin was diluted in 0.5 mg/mL alpha casein in PEM plus 1 mM ATP. The final concentration of kinesin used in these experiments was 27.5 μg/mL.
- After 10 minutes, kinesin was flowed into the flow cell by fluid exchange. The volume of the flow cell was approximately 10 μL. To ensure that all the fluid in the flow cell was exchanged properly, 20 μL of the kinesin mixture was flowed into the chamber. This was then allowed to sit for 5 minutes.
- During the various incubation steps, a new motility solution was prepared for each assay. The motility assays consisted of:
- After the 5 minute incubation with kinesin, 20 μL of the motility solution was introduced to the flow cell by fluid exchange.
- The flow cell was either sealed with nail polish or cellophane dependent on which assay was to be performed.
After sealing the flow cell, the slide was immediately placed on the microscope for observation. For a detailed description of why I used these parameters, please see Chapter 2. Briefly, the camera was set take images as follows.
- 600 total frames which was approximately 2 minutes total exposure.
- EMCCD gain of 150.
- 100 ms single exposure.
- Frames were taken at 5 frames/second.
Please see Chapter 2's Data analysis section for a detailed description of how the data was analyzed.