Cytoplasmic dynein and KASH5: partners in fertility

The birth of a healthy baby is the culmination of many complex processes, the first of which is the production of normal egg and sperm. This needs a specialised type of cell division, called meiosis. In most cells in our body, we have two copies of each chromosome: one each from our mother and father. Early during the first meiotic cell division, the two copies pair up in a process called synapsis, and swap regions of their DNA to give new combinations of genes. Synapsis is made much more efficient by movement of the chromosomes after they become attached to the membrane that surrounds the chromosomes, the nuclear envelope (NE). Chromosome movement is driven by a tiny motor, dynein, that walks along protein tracks in the cell pulling cargo with it. Dynein is linked to the chromosomes in meiosis by a protein complex that spans the NE, which contains a protein called KASH5. Dynein at the NE is also needed right after fertilization, when it transports the nucleus of the egg towards the sperm nucleus to allow their DNA to mix before the first division of the embryo. It is not known yet how dynein binds to the NE in this situation, but we predict that KASH5 does this job too.

In this project, we will analyse in depth the mechanism and regulation of the dynein-KASH5 interaction, and see if it is also needed for pronuclear migration. Dynein is a very complex protein that is made up of 6 different subunits. Our preliminary work strongly suggests that KASH5 binds to dynein's light intermediate chains (LICs). We will work out which parts of KASH5 bind to which bit of the LIC, and how this interaction is regulated during the cell cycle. Very recent work has shown that dynein is activated by forming a stable complex with two other protein components: dynactin, and one of several adaptor proteins. We think that KASH5 acts as an adaptor protein too. We also predict that the LICs play a crucial role in assembling these active dynein complexes. We will test those ideas here.

Cytoplasmic dynein and the nuclear membrane protein KASH5 work together to drive chromosome movement in meiosis I, which is crucial for homologous chromosome pairing and meiotic recombination. Dynein is activated by adaptor proteins that promote the formation of a stable tripartite dynein-dynactin-adaptor complex, and some of these adaptors bind dynein's light intermediate chain (LIC). The LICs are located at a key position between dynein's motor domain and the dynein tail-adaptor-dynactin complex, and we propose that they are perfectly placed to play a role in tripartite complex assembly, function and regulation.

We will determine if KASH5 is a new member of this adaptor protein class. We will map the domains of KASH5 and LIC that are required for this interaction and establish whether the same LIC regions bind to the adaptor proteins BicD2 and Rab11-FIP3. We will also test the hypothesis that an interaction with the LIC is a previously unrecognised requirement for tripartite complex assembly in general, by investigating whether stable complexes form between dynein, dynactin and KASH5, BicD2 or Rab11-FIP3 after LIC depletion.

Dynein at the nuclear envelope (NE) is needed after fertilization, to pull male and female pronuclei together. We propose that KASH5 is responsible for this recruitment in mammals. We will determine if KASH5 is present on pronuclei, and test if the dynein recruited to the NE by KASH5 can drive nuclear migration in vitro. Finally, since KASH5's known functions occur when the NE is intact, a key question is what happens when the NE disassembles during prometaphase, when the LICs become highly phosphorylated. We will analyse KASH5-dynein interactions in metaphase-arrested cell extracts, and test the effects of LIC phosphorylation.

This work will provide in-depth understanding of the mechanism, function and regulation of dynein's interaction with an important cargo adaptor, KASH5.

This project is focussed on obtaining better understanding of two fundamental biological processes that rely on cytoplasmic dynein function: meiotic chromosome pairing, and pronuclear migration after fertlization. These topics fit under the "Healthy ageing across the lifecourse" strategic aim, which considers the whole lifespan from conception to old age. Clearly, this basic research is of relevance to the area of reproductive biology and assisted reproductive technology, and has the potential to lead to new understanding of pronuclear migration in intracytoplasmic sperm injection. Furthermore, developing methods to disrupt the dynein-KASH5 interaction might be of use in the identification of new contraceptive targets. There are also potential long-term benefits to health and bionanotechnology from the basic scientific knowledge that will be obtained through this research.

If KASH5 turns out to be a new adaptor protein that promotes the formation of active dynein-dynactin complexes, then this opens up possible routes for stimulating dynein function. Dynein has a wide range of other potential pharmaceutical applications e.g. successful gene therapy requires fast active transport to the nucleus to deliver the DNA. Dynein transport is used naturally by viruses to avoid the body's defenses and could be targeted for improved synthetic carriers. The medical industry may be interested in ways to treat hereditary diseases associated with cytoplasmic dynein dysfunction e.g. retinitis pigmentosa, Lissencephaly (smooth brain disease), motor neuron disease. In addition, the findings from this research may be significant for the bionanotechnology field, if they enable the development of more robust motors for nanotechnological applications.

The function of microtubule motors is a topic that will be of general interest to the public and to schools, mainly because of the immediate visual impact of the work. The generation of a model that shows how such a complex molecular machine works should help interest both groups in fundamental biological processes. In addition, it may help to counter fear of nanotechnology.

The PDRA will benefit from excellent training in a wide range of cellular, molecular and biochemical approaches. This will include sophisticated light microscopy techniques. In addition, the PDRA will become skilled in protein purification. The PDRA and technician (Quentin Roebuck) will benefit from training in a wide range of translational skills via Faculty of Life Sciences staff training programmes, and generally by day-to-day activities on the project. The PDRA will also develop their writing and presentation skills, both for a scientific and a lay audience. The generation of a highly trained light microscopist/cell biologist will enhance the UK skills base.

This project will further develop the collaboration between V. Allan and B. Burke in the A*STAR Institute of Medical Biology in Singapore. The regular visits to Singapore will provide an excellent opportunity for the PDRA to develop communication skills, to learn new techniques while there, and make use of facilities not available in Manchester. The trips will also allow discussions focussed on developing new collaborations between VA and academic researchers or companies. This application therefore fits under the International Partnerships responsive mode priority.