User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/03/12/Kinesin & Microtubules

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Paper

I'm not sure of copyright laws so instead of posting the paper, I will link to it.

Nanomechanics of Microtubules

Basics

SJK 00:13, 13 March 2009 (EDT)
00:13, 13 March 2009 (EDT)This is a great page!  You've really nailed your introduction to microtubules.  Great drawing and really great understanding for day one.  You even noted that your drawing is idealized, which I think people forget sometimes.  Here's the paper by Haiqing Liu when she was in George Bachand's lab at Sandia: http://www3.interscience.wiley.com/journal/121544300/abstract (it actually made the cover!). In that paper (I was a middle-author), we talked a bit about how the way the MTs are polymerized dictates how many protofilaments there are, and this determines things about helicity, which affects the "circles" formation. (I didn't write any of the stuff about protofilament number / helicity)
00:13, 13 March 2009 (EDT)
This is a great page! You've really nailed your introduction to microtubules. Great drawing and really great understanding for day one. You even noted that your drawing is idealized, which I think people forget sometimes. Here's the paper by Haiqing Liu when she was in George Bachand's lab at Sandia: http://www3.interscience.wiley.com/journal/121544300/abstract (it actually made the cover!). In that paper (I was a middle-author), we talked a bit about how the way the MTs are polymerized dictates how many protofilaments there are, and this determines things about helicity, which affects the "circles" formation. (I didn't write any of the stuff about protofilament number / helicity)

Before I go anywhere with this paper, I need to review the basics of microtubules. This is my introduction to these items so hopefully I will get some things correct about them.

The first thing to talk about is the structure of microtubules. Microtubules are made up of heterodimer subunits. The sub-subunits are called α- and β- tubulin. These sub-subunits combine together and are called tubulin. Yep. Pretty redundant and ridiculous terminology, thanks biology! These sub units form what is called a protofilament of which microtubules are made out of.

Okay. This is ridiculous. Especially the usage of the words heterodimer, redundant usage of tubulin, and protofilaments. A picture is worth a thousand words and here goes one.

I will order the terminology going from smallest to largest.

  • Sub-subunits
    • α- and β- tubulin. The green or red balls in the picture.
  • Subunits
    • Tubulin = α-tubulin + β-tubulin
  • Protofilament
    • A single chain of tubulin running the length of a microtubule that is made of α- and β- tubulin. The orange balls in the image. Please note that the orange balls are still made up of α-tubulin and β-tubulin. I just changed their color so that I could differentiate them in the picture.
  • Microtubule
    • This is made up of 13 protofilaments of tubulin that form a hollow cylinder. The outer dimension of microtubules is about 25 nm and the inner dimension is about 15 nm.

I should note that my picture is not to scale and real microtubules are not as regular as what I have drawn. I drew the least amount of packing possible for the α-tubulin and β-tubulin subunits. I think in nature they must pack together more efficiently. I might make a better picture later but for now it works for me. I'm sure the packing geometry is simple once you know the size of each tubulin.

What's nice about my picture is that it shows the microtubule as "polar". In this case, what biologist mean by polar is that one end of the microtubule is capped with α-tubulin and the other end is capped with β-tubulin. Microtubules do form in this regular pattern of αβαβαβ sub-subunits. Each protofilament also aligns themselves in the same orientation. Biologists have termed each end of a microtubule as a slow growing (or -) end and a fast growing (or +) end. The fast\+ end is capped with β-tubulin and the slow\- end is capped with α-tubulin.

Review

  • They describe that microtubules perform vital functions in cells. Including,
    • Morphology
    • Building blocks for cilia and glagella
    • Tracks for molecular motors
  • They also say that the dynamics of microtubules in cells play a role in,
    • Cell division
    • Motility

The goal: Measure Young's and the shear moduli in vitro.

  • They note that typical experiments have to functionalize AFM tips and substrates while their setup does not.
  • They note that previous experiments measured the bending stiffness of microtubules.
  • They note that protofilaments are connected to each other by the β-tubulin sub-subunit.
  • They define the quantities they are looking for as stretching protofilaments (Young's modulus) and sliding between protofilaments (shear modulus).
  • They also relate the bending modulus to the experiments they do by saying that a large bending radius does not cause that much shear, thus they use the Young modulus. However, a small bending radius causes shear and they find the shear modulus.
  • To measure the bending modulus, which gives them the shear modulus, they used a substrate that has 200 nm regular hole patterned on to it. A microtubule would fine its way over a hole and then they would come in with an AFM tip to bend the microtubule into the hole. They used glutaraldehyde to fix the microtubules to the substrate

Take home

  • They found that indeed microtubules are anisotropic and that the bond between α-tubulin and β-tubulin within a protofilament is stronger than the bond between adjacent protofilaments.
  • The bond strength between protofilaments is also highly dependent on temperature. If it's too hot, they break easier.
  • One can cause changes in the proto-protofilament interactions by changing the salt concentration. Especially with Mg2+ and Zn2+. You can also change it by changing the pH.
    • Steve Koch 00:10, 13 March 2009 (EDT): This is interesting. Some colleagues at Sandia studies the effects of divalent cations on microtubule stability and kinesin activity (in presence of MTs). This will be related to things we want to do (osmolytes etc.). Ca++ seems to particularly disrupt MTs and kinesin-MT activity. (I'm being lazy by saying kinesin-MT activity. The long version is: "microtubule-stimulated kinesin ATPase activity" which would be the ensemble measurement of kinesin activity. In the single-molecule world (or few molecules), we don't look at ATPase (usually) but instead "motility" or things like microtubule shuttle velocity, which is related to ATPase activity.)
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