User:Andy Maloney/Introduction to kinesin and microtubules

Purpose

This page describes what kinesin and microtubules are. It gives a general discussion of why it is important to study this system.

This is the introduction to 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 by using the provided link below.

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Introduction

Figure 1: Graphical depiction of a gliding motility assay. The yellow blobs are casein, the green molecules are kinesin and the orange cylinder is a microtubule. The drawing is not to scale.

Kinesin-1, hereafter called kinesin, and microtubules are cellular components that are vital for life. Kinesin is a motor protein that shuttles cargo from one area of a cell to another. It does so by traveling along microtubules. Microtubules can be thought of as roads designed to allow motor proteins to undergo active transport in a cell.

In this chapter, I will discuss what a kinesin molecule is and why we study it. I will also introduce what microtubules are, and what they are made up of. In the first chapter to this dissertation, I will discuss how to design and implement a gliding motility assay in order to investigate the kinesin and microtubule system. Gliding motility assays can be thought of as microtubule crowd surfing on a layer of kinesin, see Figure 1. Figure 1 also depicts generally how kinesin is supported by the passivation layer of casein. Chapter 2 discusses observations that indicate that the kinesin and microtubule system is affected by the type of passivation used. Chapter 2 also discusses the importance of temperature stabilization and how without it, obtaining stable gliding speeds are not achievable.

Figure 1 does not show the water molecules that are in the system when investigating the gliding motility assay. If I was to depict the water molecules in this picture, then you would not be able to see any of the proteins since there would be a lot of water molecule obscuring the proteins. Water is an important component in biomolecular interactions and plays a significant role in the microtubule and kinesin system. Chapter 3 discusses the results of some experiments I did that involved changing the isotope of water and measuring gliding motility speeds. It also discusses future experimental work that will allow further investigation to the importance of water interactions in the kinesin and microtubule experiment.

Finally, Chapter 4 discusses open science. This dissertation and all the notebook entries associated with its research was done openly and in the public domain. I discuss my experiences with open science and some success stories from participating in open science.

Kinesin

Figure 2: Cartoon of a kinesin molecule. This is our mascot known as Kiney. The motor domains are depicted as Kiney's feet. The motor linker links the motor domains together. The stalk or coiled coil is depicted as Kiney's torso and the cargo binding domains are depicted as Kiney's eyebrows.

Kinesin is a dimeric motor protein that can be thought of as having four distinct areas to the molecule, see Figure 2. Figure 2 is a cartoon of a kinesin molecule and is also Koch Lab's kinesin mascot called Kiney. The first major component to the kinesin molecule is the motor domains also known as heads. The motor domains are depicted as Kiney's feet. They are where chemical energy (in the form of ATP) is converted to mechanical work through hydrolysis. This statement is a very interesting one which is not fully understood and is a major area of study with the kinesin molecule (Cross 2000, Coppin 1997).

Kinesin walks along a microtubule in much the same way humans walk: we place one foot in front of the other in order to take a step. The difference between how we walk and how kinesin manages to walk along a microtubule is that we have a brain that sends signals to our legs in order for them to take a step. Kinesin does not have a central processing center and thus needs a different method for communication between the two motor domains to signal steps. This communication comes from the linkage of the two motor domains via the motor linker which is also known in the literature as the neck linker. The motor linker can be thought of as a stretchy polymer of amino acids that connects the two motor domains together. The stretchiness of the polymer is a key component to how kinesin sends signals to each of the motor domains (Guydosh 2006, Yildiz 2008). The trailing motor domain of a kinesin takes a step by first docking the motor linker. This docking results in strain along the motor linker that is a signal for the leading motor domain that the trailing motor is going to take a step. These steps are gated by nucleotide presence and nucleotide states within the motor (Herskowitz 2010).

Figure 2 shows the stalk or coiled coil region of kinesin as Kiney's torso. This is where the two protein chains of kinesin coil around each other and terminate in the cargo binding domain depicted as Kiney's eyebrows. Kinesin is a motor protein that transports cellular cargo (Duncan 2006, Muresan 2000, Nakata 2003). It travels along microtubules in one direction taking 8 nm steps (Svoboda 1993) using one ATP molecule per step (Coy 1999). Kinesin actively transports cargo from one area of the cell to another and can do so by traveling along the microtubule at about 1 μm/s (Herskowitz 2010). This is essential for cellular function. For instance, sciatic nerves can be nearly a meter long. All components generated in cells are done in their nucleus. If diffusion was the only form of transportation for these items, it would take approximately 30 years for proteins made in the nucleus to be transported to the end of the nerve a meter away. If kinesin were to transport the same items, it would take about 2 weeks.

If the kinesin microtubule system is affected by mutations in kinesin, then this can lead to a variety of diseases (Goldstein 2001, Hurd 1996, Goshima 2003). One such disease causes loss of motor function due to accumulation of cellular cargo transported by kinesin which is lethal. Other mutations that affect humans include Alzheimerʼs disease (Stokin 2005).

There are several types of kinesin molecules as well as many different types of motor proteins (Sack 1999). This thesis attempts to illuminate experimental procedures necessary in order to obtain good signal to noise data in order to investigate the kinesin and microtubule system.

Microtubules

Microtubules are one type of road produced in cells that kinesin and other motor proteins walk on. They are polymers made up of subunits called tubulin. Tubulin in turn is made up of two different proteins called α-tubulin and β-tubulin. α-tubulin and β-tubulin combine together to for a single tubulin subunit via a GTP molecule. The bond between α-tubulin and β-tubulin is fairly robust and does not

References

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