Transport to the centre of the cell: discovering dynein's stepping mechanism by cryo-electron microscopy

The development of every human body starts from the single cell formed by fertilization of an egg cell by a sperm cell. The cell divides into two, four, eight and so on, ultimately to make the hugely complex human body. In each cell division the DNA and other cell components must be separated equally into the daughter cells, and this is achieved by a special scaffold framework called the protein cytoskeleton. In this process, there must be a source of force to move the DNA and cellular components into the correct locations in the daughter cells. The force is supplied by motor proteins, which interact both with the cytoskeletal proteins and with the DNA etc, and they act like tiny railway engines carrying their cargoes along the cytoskeleton tracks. During the day-to-day life of the cell, these motor proteins act as transporters between the cell surface and its nucleus and between different structures within the cell.
The length of a single human nerve cell can be more than a metre from the tip of the toe to the spinal cord within the backbone. The proteins necessary to maintain nerve cells can be made only in the cell body near the nucleus, not at the tip of the nerve in the toe. So proteins needed in the toe region must be transported there. Some proteins and other cargoes are transported back to the cell body. Abnormal function of the motor proteins in nerves can cause neurodegenerative diseases. Moreover, HIV, Herpes and other viruses can infect the body through a surface of the neuronal cell and are then transported into the cell nucleus along that one metre distance, where they can then wait to cause disease later on. This movement is also driven by the motor proteins, which move along the cytoskeletal transport network.
If we understood the principle behind the movement of the motor proteins, we would gain a new target for drugs to prevent virus infection and to prevent the rapid cell division in cancer cells. We already know that the motor protein, dynein is involved in all the movements we have mentioned, but we don't know how it works, so we don't know how to regulate it.
Dynein is a motor protein that uses a high energy fuel molecule called ATP to generate force on cytoskeletal tracks called microtubules. There are two major classes of dyneins. One is for transporting cargoes from the periphery of the cell towards the centre, and is the motor that gets hi-jacked by viruses. It also is needed for cell division to work properly. The other dyneins are what make the tails of sperm cells wiggle so that they can swim to fertilise the egg. So dynein is important for many vital functions of the body.
The transporter dynein consists of a pair of identical motor domains, which are joined together by a complicated tail. To understand how the motor works, we make just the dynein motor domain with a tag that holds two motors together. This mini-dynein walks along microtubules using ATP fuel, but because it has no tail, it can't carry cargo.
To understand how dynein walks on microtubules, we will study the structure of this mini-dynein by flash freezing it while it is walking and then viewing the individual molecules using a special electron microscope. By looking at many thousands of molecules we will see the different structures dynein adopts as it walks. Our study is helped by special computer software that lets us see the 3D structure more clearly.

Dynein belongs to the AAA+ protein family. It is a molecular motor, which converts chemical energy from ATP hydrolysis into mechanical movement along microtubules. Dynein is an essential protein from yeast to human. There are two kinds of dynein, cytoplasmic dynein and axonemal dynein. Axonemal dynein is responsible for movement of cilia and flagella. Cytoplasmic dynein transports cargoes such as mRNA, membrane vesicles, viruses and mitochondria towards the minus end of the polar microtubule. In general that is transport towards the centre of the cell. Conformational change in isolated dynein has been studied between the absence of nucleotides and the presence of ADP and ADP-vanadate. The linker of the dynein switches positions between unprimed and primed conformations when ADP-vanadate binds. This linker swing is thought to be the mechanism of dynein movement. The few studies of dynein structure on microtubules are consistent with the linker swing hypothesis.
Cytoplasmic dynein walks on microtubules as a dimer in the presence of ATP. However, there is no structural study of cytoplasmic dynein on microtubules in the presence of ATP even though this is the physiologically important structure. This is because in the presence of ATP, dynein readily falls off the microtubule. The aim of this research is to understand the structural mechanism that cytoplasmic dynein uses to walk on microtubules. To do so, we will use flash-freezing and cryo-electron microscopy to determine the range of structures adopted by the walking molecules on microtubules in the presence of ATP. By using tilted specimens we will gain 3D structural information. The outcome of this study could be potentially useful to derive novel therapies against cancer and neurodegenerative disease and virus infections such as HIV.

Potential Beneficiaries
Dynein dysfunction causes a number of human diseases including infertility, situs inversus viscerum and neurodegenerative disease and cancer. Therefore, any therapy of the above diseases,developing as a consequence of dynein research, could be beneficial to the British society and economy in the long run through pharmaceutical industry and health and well-being. However, the connection from dynein academic research to industry and the wider public in general is still immature at this moment. In order to reinforce the connection, we made an impact plan through the following series of outreach activities.

Academic community and industry
We will achieve academic impact by presenting our research results to the academic community at three different major international conferences: an American Biophysical society meeting, an American Society for Cell Biology meeting; and a Gordon Research Conference on motor proteins. We will also achieve long term impact by publishing two papers in high impact open access journals. We previously published in Nature, Cell, and Nature Structural and Molecular Biology. One of our previous dynein studies has been included in the internationally famous cell biology textbook, "Molecular Biology of the Cell", fifth edition by Bruce Alberts et al. This textbook is widely used for undergraduate education. Therefore, our research has contributed to the academic community including international cell biology education.
The Astbury Centre for Structural Molecular Biology at University of Leeds is vigorously developing links with industry to facilitate communication both ways between industry and academics. We have been involved in this activity and will continue to engage in it including presenting results from this project.

Wider public in general
We believe there is a wide appetite among the general public to better understand the natural world. When our paper on dynein came out in Nature in 2003, our conclusions were featured in the national newspapers The Sun and Financial Times and the local newspaper Yorkshire Post and the BBC news website. We will contact the Press Office when we succeed in publishing high impact papers from this project.
The annual Leeds Festival of Science is a three-week outreach activity for school pupils. The PI was involved in this event in 2010, and we will incorporate dynein into our participation.
We also will visit local schools several times during the grant period to alert pupils to the interest and importance of motor proteins. Our Faculty of Biological Sciences has a program for local school visits. The investigators are all registered on the program.
The Co-I gave a public lecture in 2005 in Kobe, Japan. The RCo-I demonstrated research in2002, 2005 and 2006 for the general public audiences and high school students.
We have productively collaborated with researchers in Japan. Our research has the social benefit of fostering good UK-Japan international relationships.

Capability
Presenting the work and writing publications will be undertaken as a team effort by the PI, Co-I and RCo-I, with our collaborators. Press releases will be written with the help of our Press Offices. We have a good track record of public lecture and explanation of research to the general public and contacting industrialists and we plan to continue and enlarge these activities. The RCo-I has attended training courses run by both the Faculty of Biological Sciences and the Universityof Leeds on knowledge exchange and impact, and will attend any new courses that will enable us to adopt novel approaches in our outreach activities.

Resource
For the benefit of school children, we will engage in the annual Leeds Festival of Science event, and have requested funds for posters and display materials in our costs. We have also requested funds for participation in conferences, and to publish in highest impact, open access journals.