User:Andy Maloney/Surface passivation effects on kinesin and microtubules

Purpose

This page describes the experiment where I investigated gliding speed variations of microtubules using different surface passivation schemes.

This is the second 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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Introduction

Figure 1: Image of our mascot "Kiney" at the microscope where I perform experiments. The plush doll form of Kiney was made by Child's Own Studios.

Both kinesin and microtubules are vital components of eukaryotic cells. Kinesin is used as a vehicle to shuttle cellular items from one part of the cell to another and microtubules are the roads that kinesin travel on. Kinesin walks along microtubules in one direction and does so by converting chemical energy, in the form of ATP hydrolysis, to linear motion^(1-5). Observations of this system are typically carried out in two manners: the gliding motility assay or with an optical trap^(6-8). The gliding motility assay^(9-11) is the experimental procedure of interest for this study and will be explained in greater detail below.

In the gliding motility assay, motility is sustained by first passivating the glass microscope slides. Passivation is done to prevent kinesin’s motor domains from becoming inactive when interacting with untreated glass. It is not understood how or why kinesin motor domains become inactive on untreated glass but they do. Passivation of glass can be done with bovine serum albumin (BSA)^(9-11), bovine casein^(12-16), a lot of kinesin⁽¹⁷⁾, or other chemical compositions⁽¹⁸⁾. Bovine casein is the surface blocker of choice by many experimenters mainly because it works well at passivation and is inexpensive. 500 g of whole bovine casein costs about $30 at the time of this writing. Typical assays will use 10 - 50 μg of casein at a time. This means that the 500 g stock of bovine casein will outlast a graduate student's career and if stored properly, possibly a PI's. Casein is a globular protein that does not have a known crystal structure⁽¹⁷⁾. Bovine casein is comprised of four major subgroups: α_s1, α_s2, β, and κ. Depending on the mammal the caseins come from, there exists different ratios of these globular constituents. For instance, bovine casein contains α_s1 + α_s2 > β > κ and human casein contains β > κ with only trace amounts of α_s1 casein^(19,20). Each species has a finely tuned milk for their neonates as casein is a vehicle in milk for delivering calcium phosphate and amino acids to them.

How casein passivates glass surfaces in order to support kinesin for the gliding motility assay is still not very well understood but, some work has been done to try and understand it. Ozeki et al. showed that two layers of casein form on the glass surface to help support kinesin for motility⁽¹²⁾. Verma et al.⁽¹⁷⁾ showed that the number of microtubules that landed on the kinesin surface was affected by the casein passivation. Hancock and Howard also showed that the number of microtubules that landed on the kinesin surface was dependent on the number of motor proteins adhered to the glass slide⁽²¹⁾.

These studies show that the type of casein used in an assay affects the kinesin and microtubule system. One way to measure this affect is to observe the speed at which microtubules glide. This study aims to show that there are subtle differences in the speeds at which microtubules will glide at and I will offer some of my own ideas as to why I think these differences exist.

Methods and materials

The experimental procedure relies heavily on the methods and materials already discussed in Chapter 1. Here I will outline the subtle differences in the generic gliding motility assay to the surface passivation assay. This assay tries to gain more insight into how the affect of kinesin adhering to the passivation layer on the glass substrate changes the way kinesin interacts with microtubules.

Temperature stabilization

Figure 2: Image of the IX71 Olympus microscope used for experiments.

Experiments were performed on an Olympus IX71 inverted microscope. The objective used was a 60x 1.42NA oil immersion PlanApo objective from Olympus. Fluorescence excitation of the rhodamine fluorophores attached to the microtubules was done with a 100 W mercury lamp, also from Olympus. The lamp was attached directly to the microscope and filtered for rhodamine's excitation and emission using Chroma's TRITC filter set. The mercury lamp was attenuated by 94% to prevent heating of the sample and to prevent opticution of the microtubules. Image sequences were taken with an Andor Luca S EMCCD camera controlled by custom LabVIEW software.

My initial experiments on the microscope gave me similar data to what is shown in Figure 3. All the data would have an initial increase in speed when I first put it on the microscope and then, over time it would taper off but still increase. Removing the first 5 data points from the curves in Figure 3 gave me errors for average speeds of microtubules in range with the literature. The speed increase over time bugged both me and Dr. Koch but we didn't know what was causing it. Dr. Koch suggested that I present this information at BPS 2010 and see what people thought about it.

Mixed in with the whole speed increase over time issue, I became obsessed with why we would use milk proteins (caseins) to coat the glass in order to sustain motility. After reading the paper by Verma et.al.⁽¹⁷⁾ and the book by Fox and McSweeny^AA, I somehow convinced myself that there would have to be a speed change when using different passivation proteins. So, I began looking at caseins and how they affected how kinesin would interact with microtubules. Much to my surprise, I in fact did see a speed change when using different passivation proteins. Figure 3 shows a remarkable change in speed for different caseins used.

Figure 3: Graph showing three experiments using different passivation schemes. Note that each individual trace continuously increases in speed over time.

When I did present this information at BPS, Stefan Diez suggested that the reason why I saw an increase in speed over time for single assays was due to a temperature effect. At the time, I thought he was out of his mind because there was nothing attached to the microscope that could be heating it up. Or, so I thought. I continued to take data and tried other experiments with osmolytes and water isotopes, and I didn't think of the temperature issue very much since I had taken data that proved my hypothesis that casein passivation affected gliding speeds.

I presented more of the same data with data showing speed changes due to water isotopes and osmolytes at a conference in Santa Fe. Erik Schaeffer was attending the conference and he again told me that there was more than likely a temperature issue going on with the speed increases I saw. I again wasn't very convinced until he talked about his microscope setup. He told me that he had his objective temperature stabilized with some flexible heating elements and was able to achieve millikelvin precision with his setup. He also told me of a story that is fascinating for a graduate student to know. Erik mentioned that with his setup, he could tell rather precisely when and if the graduate students in the lab turned on the computer monitor next their tweezers with the stabilized objective. This story blew me away and I of course didn't think it possible.

After the conference, Steve and I did a few experiments to see if in fact the microscope was heating up because of the mercury lamp that was attached to it. It turns out that the mercury lamp does heat up the microscope on a time scale of something like 12 hours and it was indeed affecting my experiments.

Figure 4: Graph showing the three experiments from Figure 3 plotted with respect to the time of day they were performed at.

Thankfully, I did the three experiments in Figure 3 all on the same day. Another big thanks needs to go to the programming prowess of Larry Herskowitz and Steve Koch for time stamping all the data gathered because it allowed me to plot the assays against each other with absolute time. As can be seen in Figure 4, it turns out that the mercury lamp heated up the microscope considerably over the course of my data taking. I should emphasis that Figure 3 and Figure 4 are the same data and the only reason why there is an increase in speed is from the temperature increase of the microscope.

Of course, this finding was unfortunate because it basically said that the stark differences in gliding speeds was solely due to temperature affects and not the chemistry of passivation. Nonetheless, I took this opportunity to build a similar system to what Erik Schaeffer uses in his lab to stabilize the temperature of the microscope objective. As can be seen in Figure 5, with a stable temperature, speed measurements can be recorded with as little as ±4 nm/s errors. This is a feat all to itself since average speeds in the literature can quote errors as large as 50 nm/s Cite. Even with the temperature stabilization, I was still getting the large increase in speed observed in my earlier experiments that did not have temperature stabilization. This turns out to be because I prepare samples at room temperature and the microscope objective is set above room temperature in order to maintain a stable temperature.

Figure 5: Graph showing speed values after temperature stabilization.

A detailed description of the build can be found in an Instructables post and a brief description can be found in a previous chapter. This design can maintain a temperature to ±0.1°C. There are very few studies that indicate whether or not observation of the gliding motility assay was done with temperature stabilization or not. It has been shown that temperature does play a crucial role in obtaining stable data^(10,11) in a few studies as well as the ones I have done.

Flow cells and solutions

Preparation of the tools necessary to conduct this experiment is paramount. Detailed descriptions of these tools are outlined in a previous chapter. Briefly, in order to conduct this experiment, I required the following.

• 10x PEM buffer
• Antifade system
• Tubulin
• PEM-A
• Surface passivation chemicals

Surface passivation chemicals

This experiment relies on the different bovine caseins to investigate speed variations of microtubules in the gliding motility assay. In the temperature stabilization section of this chapter, I thought that I was seeing huge difference in speed with the different caseins used. It turns out that the speed variations are a bit more subtle but, they definitely do exist. A mixture of the α_s1- and α_s2-caseins purified to 70%, β-casein purified to 98%, and κ-casein purified to 70% were purchased from Sigma (C6780, C6905, and C0406 respectively). Each casein component was reconstituted in PEM under constant stirring. α-casein took approximately 60-80 minutes of constant stirring before it went into solution, β-casein took approximately 30-40 minutes, and κ-casein required 15-20 minutes, all at room temperature. Whole casein (Sigma C7078) however, did not go into solution easily without the addition of heat. Whole casein is not susceptible to thermal denaturation and is not affected by moderate heating Cite. Heating PEM to 60˚ - 80˚C while stirring in whole casein, allowed the whole casein to dissolve into PEM while a condenser prevented evaporation of the buffer. All casein solutions were reconstituted to 1.0 mg/mL in PEM. After all the casein was dissolved in solution, it was stored at 4˚C in convenient aliquots with no additional filtering. I did not investigate the solubility of the caseins in a solution of PEM. Casein solubility is a complicated function of pH Cite, temperature Cite, calcium concentration Cite, and ionic strength Cite. Solubility is even dependent on the concentration of other casein constituents in solution Cite. <html> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> </html>

References

1. Carter, NJ, & Cross, RA (2005). Mechanics of the kinesin step. Nature, 435(7040),308-12. doi: 10.1038/nature03528
2. Vale, RD, & Fletterick, RJ(1997). The design plan of kinesin motors. Annual review of cell and developmental biology, 13, 745-77. doi: 10.1146/annurev.cellbio.13.1.745.
3. Hua, W, Young, E, Fleming, M, & Gelles, J (1997). Coupling of kinesin steps to ATP hydrolysis. Nature, 388, 390-393.
4. Vale, R, Reese, T, & Sheetz, M (1985). Identification of a novel force generating protein, kinesin, involved in microtubule-based motility. Cell, 42(1), 39-50. doi: 10.1016/j.bbi.2008.05.010.
5. Goldstein, LSB, & Philp, AV (1999). The road less traveled: emerging principles of kinesin motor utilization. Annual review of cell and developmental biology, 15, 141-183.
6. Block, SM, Goldstein, LSB, & Schnapp, BJ (1990). Bead movement by single kinesin molecules studied with optical tweezers. Nature, 348, 348-352. doi: 10.1038/348348a0
7. Yildiz, A, Tomishige, M, Vale, RD, & Selvin, PR (2004). Kinesin walks hand-over-hand. Science (New York, N.Y.), 303(5658), 676-8. doi: 10.1126/science.1093753.
8. Gelles, J, Schnapp, BJ, & Sheetz, MP (1988). Tracking kinesin-driven movements with nanometre scale precision. Nature, 331(6155), 450-3. doi: 10.1038/331450a0.
9. Böhm, KJ, Steinmetzer, P, Daniel, A, Baum, M, Vater, W, & Unger, E (1997). Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion dependent motility. Cell motility and the cytoskeleton, 37(3), 226-31. doi: 10.1002/(SICI)1097-0169(1997)37:3<226::AID-CM4>3.0.CO;2-4.
10. Böhm, KJ, Stracke, R, Baum, M, Zieren, M, & Unger, E (2000). Effect of temperature on kinesin driven microtubule gliding and kinesin ATPase activity. FEBS letters, 466(1), 59-62. doi: 10.1016/S0014-5793(99)01757-3.
11. Böhm, KJ, Stracke, R, & Unger, E (2000). Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters. Cell biology international, 24(6), 335-41. doi: 10.1006/cbir.1999.0515.
12. Ozeki, T, Verma, V, Uppalapati, M, Suzuki, Y, Nakamura, M, Catchmark, JM, et al. (2009). Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces. Biophysical journal, 96(8), 3305-18. doi: 10.1016/j.bpj.2008.12.3960.
13. Woehlke, G, Ruby, AK, Hart, CL, Ly, B, Hom-Booher, N, & Vale, RD (1997). Microtubule interaction site of the kinesin motor. Cell, 90(2), 207-16. doi: 10.1016/S0092-8674(00)80329-3.
14. Moorjani, SG, Jia, L, Jackson, TN, & Hancock, WO (2003). Lithographically Patterned Channels Spatially Segregate Kinesin Motor Activity and Effectively Guide Microtubule Movements. Nano Letters, 3(5), 633-637. doi: 10.1021/nl034001b.
15. Hess, H, Clemmens, J, Qin, D, Howard, J, & Vogel, V (2001). Light Controlled Molecular Shuttles Made from Motor Proteins Carrying Cargo on Engineered Surfaces. Nano Letters, 1(5), 235-239. doi: 10.1021/nl015521e.
16. Ray, S, Meyhöfer, E, Milligan, RA, & Howard, J (1993). Kinesin follows the microtubule’s protofilament axis. The Journal of Cell Biology, 121(5), 1083-1093. doi: 10.1083/jcb.121.5.1083.
17. Verma, V, Hancock, WO, & Catchmark, JM (2008). The role of casein in supporting the operation of surface bound kinesin. Journal of biological engineering, 2, 14. doi: 10.1186/1754-1611-2-14.
18. Howard, J., Hudspeth, A. J., & Vale, R. D. (1989). Movement of microtubules by single kinesin molecules. Nature, 342(6246), 154-158. Nature Publishing Group. doi: 10.1038/342154a0. doi: 10.1007/BF00421079.
19. Fiat, A.-M., & Jollès, P. (1989). Caseins of various origins and biologically active casein peptides and oligosaccharides: structural and physiological aspects. Molecular and cellular biochemistry, 87(1), 5-30.
20. Fox, P, & McSweeney, P (1998). Chapter 4: Milk Proteins. Dairy chemistry and biochemistry (1st ed., pp. 146-238). London: Blackie Academic & Professional.
21. Hancock, WO, & Howard, J (1998). Processivity of the motor protein kinesin requires two heads. The Journal of cell biology, 140(6), 1395-405. doi: 10.1083/jcb.140.6.1395.

Notebook entries

All of my raw observations, raw data taking, and silly ramblings are contained in my notebook entries. They are the unedited versions of this page.