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 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

In this section, I will discuss the technological hurdles required to obtain stable data and the subtle differences in the generic gliding motility assay described in Chapter 1, compared to this assay. This experiment investigated the affect on kinesin and microtubules due to the passivation layer used.

Temperature stabilization

Figure 2: Graph showing three experiments using different passivation schemes. Note that each individual trace continuously increases in speed over time.

Temperature stabilization is crucial for observing stable speeds. Without it, I observed microtubule gliding speeds increase over time. Figure 2 shows data that did not use a temperature stabilized objective and shows a sharp initial increase in speed which, over time would taper but never level off. There are three different assays in the graph, one each of: alpha casein, beta casein and whole casein. I initially thought that I observed the goal of the study; which was to observe speed changes dependent on the type of passivation used. However, what I observed was that kinesin is a very sensitive temperature probe.

I presented Figure 2 at the Biophysical Society Meeting 2010 where Stefan Diez suggested that the increase in speed over time was due to a temperature effect. I did not initially understand his comment because there was nothing attached to the microscope that could be heating the sample. So, I didn't really do anything about it. I presented more of the same data showing speed changes due to water isotopes and osmolytes at a conference in Santa Fe later that year. Erik Schaeffer attended the conference and he again told me that there was a temperature issue and that was why I saw the speed increase over time. He described to me the microscope setup used in his lab which stabilized the temperature of the objective with millikelvin precision. He told me that with this setup, he could tell rather precisely when and if a graduate student in his lab turned on the computer monitor near the microscope. This story blew me away and I of course didn't think it possible but, it most certainly is true as I found out with my experiments. If a computer monitor not even attached to the microscope could register a temperature increase on the objective, then there was a highly probable possibility that my microscope was heating up due to the mercury lamp.

Figure 3: Temperature of the microscope objective with no temperature stabilization and the mercury lamp on shining through the objective. The black curve is when the lamp was on and the red was when it was switched off.	Figure 4: Graph showing the three experiments from Figure 3 plotted with respect to the time of day they were performed at.

The temperature of the entire microscope does increase over time due to having the mercury lamp on. Figure 3 shows the temperature increase of the objective due to having the mercury lamp on and shining through it. The temperature of the objective was taken at the top, near where the sample would be and no oil was used on the objective. As can be clearly seen, the objective does reach a stable temperature, albeit 5.5 hours after turning the lamp on. This is not ideal for experiments and doesn't the effects of the placement and removal of slides nor the addition of oil on the objective.

Returning to the data taken in Figure 2, I decided to plot it with respect to the time of the day it was taken. Thankfully I took all the data in Figure 2 on the same day and when plotted with their relative time stamps from the first image taken, Figure 4, one can clearly see that there is an overall increase in speed as time goes on. As a bonus, one can also see that over the time the slide is on the objective, the speeds increase. This is due to the fact that I make my slides at room temperature and they heat up when on the objective. A plot showing the effect of temperature on the objective for turning the heating element on and off, and adding oil and removing and replacing a slide on the objective can be seen in Figure 5.

This finding I felt was unfortunate because it basically said that the large differences in gliding speeds was solely due to temperature effects and not on 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. Figure 6 also shows the large increase in speed observed in my earlier experiments for early times. This shows quite nicely that without temperature stabilization, speed measurements would not be stable.

A detailed description of the build can be found in an Instructables post and a brief description can be found in a Chapter 1. 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.

Figure 5: Temperature of the microscope objective with temperature stabilization. The initial spikes are due to turning on and off the stabilization electronics. The small spikes show when a slide is removed and a new slide at room temperature is place in its place.	Figure 6: Graph showing speed values after temperature stabilization.

Passivation and motility solutions

Preparation of the tools necessary to conduct this experiment is paramount. Detailed descriptions of these tools are outlined in Chapter 1. I like to think of the preparation for all the solutions used in an assay as my mise en place. And, just like one would never touch a fellow cook's mise en place in a restaurant, no one else but me is allowed to use my solutions. This is partly due to how finicky these assays are and how I've learned that keeping a tight control on my solutions allows me quickly replace chemicals when an assay has gone awry. For this experiment, I required the following components.

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

I have spoken about the above solutions already in Chapter 1, however, I have not spoken in detail about the passivation chemicals. Before I describe the motility assays, I will briefly talk about the passivation chemicals.

Surface passivation chemicals

This experiment used the different bovine casein constituents to investigate speed variations of microtubules in the gliding motility assay. In the temperature stabilization section of this chapter, my initial experiments saw huge differences in speed with the different caseins used. As was already mentioned above, the large speed variations were due to temperature effects and not due to the passivation chemicals. Once I stabilized the temperature of my objective, I continued to investigate if there were any variations in speed due to surface passivation. It turns out that there was a difference in speed measurements due to surface passivation, albeit a more subtle difference than what I saw initially without temperature stabilization.

Figure 7: Condenser unit attached to a solution of whole casein being mixed and heated in PEM.

To begin this experiment, I purchased a mixture of the α_s1- and α_s2-caseins purified to 70%, β-casein purified to 98%, and κ-casein purified to 70% 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 dissolving 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 dissolve into solution easily without the addition of heat. Whole casein is not susceptible to thermal denaturation and is not affected by moderate heating⁽²²⁾. 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 other casein constituents in solution^(23,24), temperature^(25-27), genetic variants⁽²⁸⁾, ionic strength and types of salts in solution⁽²⁸⁾, calcium ion concentration^(23,24,28), and pH⁽²⁹⁾. Casein solubility measurements were not performed in this study.

References

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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.