How do mutations in non-muscle myosin 2A cause bleeding disorders and other defects?

Lead Research Organisation: University of Leeds
Department Name: Sch of Molecular & Cellular Biology


All cells in the body need a 'skeleton' to maintain their shape, help them to adhere to other cells, and to move. The most highly ordered skeleton is found in muscles, where it is essential for muscles to contract and for the heart to beat. In all other cells, the skeleton (the 'cytoskeleton) is much less well ordered, and the organisation of the skeleton quickly changes in response to external signals. For example, when blood platelets are stimulated to help in blood clotting, the cytoskeleton is quickly assembled to contract the blood platelets and help them stick to the wound. This skeleton has two key protein components called actin and myosin. Myosin generates a force when it interacts with actin and this helps to control cell shape. Platelets have a specific type of myosin called non-muscle myosin 2A. This protein is found in both an inactive compact folded state which cannot interact with actin, and an active state, in which it is assembled into short filaments of about 20-30 molecules, and each of the molecules can interact with actin to contract the cell. In the inactive state, the tail of the molecule interacts with the head to keep it switched-off. Mutations in non-muscle myosin 2A are responsible for blood clotting and a range of other disorders. Based on the positions of many of the mutations, we think that they are likely to interfere with the interaction of the tail and the head of non-muscle myosin 2A and prevent proper formation of the inactive molecule. This will affect the ability of the cell to correctly regulate the assembly of non-muscle myosin 2A into filaments in response to external stimuli, and thus explain why these mutations give rise to clotting and other disorders. Our research will test this idea by finding out if the mutations disrupt the switched-off state in cells, how they affect the structure of the protein in solution, and it will investigate the interaction of the tail and the head in precise detail, to better understand how the switched off state is formed and stabilised, and how the mutations interfere with its formation.

Technical Summary

Over 80 mutations in MYH9 have been described (Human Genome Mutation Database) that collectively cause an autosomal-dominant disorder known as MYH9 disease. MYH9 encodes non-muscle myosin 2A (NM2A). In humans, the outcomes of this disease range from mild to life-threatening. Symptoms include clotting disorders in which platelets are enlarged and bleeding time is prolonged (thrombocytopaenias), aggregation of NM2A in neutrophils, cataracts, deafness and glomerulosclerosis (which can be lethal).
In platelets and other non-muscle cells, NM2A is found both as an inactive compact folded molecule, known as '10S' (based on its sedimentation coefficient), and as an active extended molecule (6S) which forms filaments of 20-30 molecules. In the 10S form, the coiled coil tail wraps around one of the motor domains, and this motor-tail interaction is important in stabilising the inactive state. Plotting the mutations onto a model of the folded molecule, together with our preliminary data, suggest that a significant number of mutations are likely to interfere with this interaction, destabilising the inactive state, which would result in premature filament formation, affecting cell contractility, and thus explain why these mutations result in MYH9 disorders. Our goal is to test this hypothesis by a combination of approaches, from investigating the effects of mutations on filament formation in cells, to investigating the effects of mutations on the secondary structure of the coiled coil, and, by using cryo-EM to obtain a detailed high resolution structure of the folded molecule, to precisely define how the tail interacts with the motor, and thus understand how mutations could disrupt this interaction.

Planned Impact

The main beneficiaries of our research will be academics and clinicians.
Our research will determine how mutations in non-muscle myosin 2A (NM2A) disturb the equilibrium between the inactive compact molecule (the '10S' form) and the filamentous, active form. This research addresses a key question in cell biology regarding the regulation of the activity of NM2A of interest to academics, and it will additionally shed light on how these mutations cause disease, of interest to clinicians. We have already engaged with the platelet group at Birmingham regarding this research. Our hypothesis is that these mutations interfere with the stability of the folded inactive molecule, increasing the likelihood that it will adopt an extended form ('6S) and form filaments prematurely, affecting the 10S/6S equilibrium, and affecting the ability of cells to control the levels of myosin incorporated into filaments. Testing this hypothesis, and in addition using cutting edge techniques including the use of cryo-EM to obtain a near-atomic structure of the 10S form of NM2A will be of broad general interest to academics. In particular the structure of the 10S form will have high general impact, and will help us understand how non-muscle and smooth muscle myosin isoforms form this specific structure, in which they are inactive.
To make sure that researchers in this area are aware of our research, we will publish our findings in journals that reach this broad audience (e.g. Nature Cell Biology, Nature Communications) as well as more specialist journals (e.g. PNAS, Blood), and present our work at national (British Society for Cell Biology, Microscopy meetings) and international meetings (American Society for Cell Biology).
Clinicians will benefit from understanding how mutations in NM2A give rise to MYH9 disorders. For example, if mutations generally result in an increase in filament formation, affecting platelet shape and contractility, this will both help them explain the disease to patients, and bring with it the potential to give patients appropriate therapies and/or develop new ones. Publishing this work in the appropriate journals to reach this audience (e.g. Blood). We also have strong links with the clinical geneticists at the Leeds Institute of Molecular Medicine at St James' hospital (including Prof. Colin Johnson, and Dr Eamonn Sheridan, with whom we have jointly funded research), and can benefit from their expertise in promoting our findings to the clinical community, as well as our contacts with platelet researchers in Leeds (Robert Ariens), and at Birmingham.


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