Regulation of myosin VI motor activity by cargo binding in vivo and in vitro

The biological cell resembles a very busy miniature factory with a large collection of dedicated protein machines working together to maintain the health and welfare of the cell. These machines have highly specific functions and are driven by chemical energy to perform a variety of different tasks. One example are the highly specialized motor proteins that can move along filament tracks in a step-by-step motion powered by the chemical energy derived from ATP hydrolysis. These motor proteins move cargo around the cell to specific locations rather like a train running along a railway network to its specific destination. There are 3 types of motors that work on either large microtubule bundles or actin filament networks, which spread like spider's webs all over the cell. Our research focuses on the molecular motor protein myosin 6, which is unique, because it is the only myosin motor that comes with a reverse gear and therefore can move in the opposite direction to all the other molecular motors along actin filaments. The cargo is hooked up to this motor with the help of a large number of specific adaptor proteins that we have recently identified. The aim of our research is to determine how the specific cargo molecules are selected and attached to the motor and how this triggers the motor to start to move to its destinations in the cell. In addition it is important to understand how this molecular machine is controlled and regulated by identifying its on/off switches, since defects in myosin 6 can cause a number of diseases including deafness and also potentially Alzheimer's disease and prostate cancer.
We thus intend to identify the mechanisms that regulate myosin 6 and its adaptor proteins for the transport of cargo to specific regions of the cell. This work will open up new areas of research that will guide future clinical studies and the development of potential diagnostic tools and possible therapeutic strategies to treat dementia and cancer.

Intracellular transport and delivery of cargo along microtubule and actin filament tracks is driven by three distinct types of motor proteins; dyneins, kinesins and myosins. The myosin superfamily of actin-based motors is directly involved in a diverse range of cellular processes that require the transport/tethering of a wide variety of different cargoes. Therefore selective cargo attachment is the key to understanding the activation, regulation and function of motor proteins within different cellular pathways. Our work focuses on myosin VI, a unique reverse directed myosin motor protein which functions in the major membrane trafficking pathways in the cell including cargo sorting and vesicle transport in the endocytic and secretory pathways.
In this proposal we plan to investigate how cargo attaches to and regulates the functions of myosin VI and develop assay systems to analyse the movement of myosin VI along actin filaments in complex with cargo in vitro and in vivo. These experiments using wildtype myosin VI and mutations identified in patients with hearing defects will allow us to gain novel insights into the mechanism and regulation of this unique myosin. Thus we aim to characterise myosin VI and its adaptor/cargo binding at the molecular and cellular levels using selected mutants and biochemical, EM, NMR, steady-state and transient kinetic methods together with single molecule experiments using optical tweezers and TIRF microscopy and a combination of functional binding assays, high resolution microscopy and live cell imaging. More specifically we aim to establish whether myosin VI exists in an auto-inhibitory folded state and whether cargo attachment activates and regulates its motor properties. We will also measure the motor properties of myosin VI when it associates with different cargo adaptors in vitro and develop novel assay systems to determine myosin VI-dependent movement of cargo along actin filament bundles in microvilli in live cells.

Who will benefit from this research?
The public and wider academic community will benefit from the new information on crucial processes in basic cell biology and motor protein function; such as how do cells move, how within cells do components/cargo move around and how do they know where to go? By studying these "biomotors", we will increase our knowledge on the mechanochemical properties and regulation of these fascinating protein machines that are of potential interest in biology, material sciences and chemistry. This work may create potential spin-offs for sensors, electronics, bio-engineering and importantly in medicine. Of particular importance and interest for the general public is the increase in our knowledge about proteins involved in diseases such as deafness, prostate cancer and dementia. These devastating diseases affect a high proportion of our aging population in the UK (and world-wide) and place a heavy burden on our health budget. Myosins are drug targets and we are currently testing new small molecule compounds designed to target specific classes of myosins in our cellular assay systems. Thus sectors of the pharmaceutical industry that develop drugs to treat cancer, deafness and neurological disorders will be interested in the new discoveries and knowledge generated by this work. In the long term, patients suffering from these diseases may also benefit and thus there will be a crucial positive impact on improving health, wellbeing and wealth in the UK.

How might they benefit from this research?
1. Industry and Bionanotechnology: This work will lead to a better understanding of the regulation of molecular motor proteins, which potentially can form the basis for single molecule devices to be developed in the field of bionanotechnology. It will help to establish regulatory and control mechanisms in the cellular environment, but will also enable one to use these principles for technological gain beyond their original setting. Our work will produce information about how to assemble and regulate motor/cargo complexes that can potentially perform controllable tasks involving mechanical movement at the nanoscopic scale and thus could be important for the development of miniature devices in the biomedical industry, a commercial development of bionanotechnology.
Myosin VI is important because it is the only motor that moves towards the minus end of actin filaments and therefore has unique cellular functions. Thus the basic research outlined in this proposal is likely to have a direct impact upon future strategies for treatment of a number of devastating diseases and is essential for the setting up of comprehensive clinical trials.
2. Health care professionals and patients will benefit from work undertaken in this study. Although this proposal focuses on basic research to determine how a motor protein functions at the molecular level and how motor activity is regulated, this work will also advance our understanding how this myosin is linked to inherited forms of deafness and neurodegeneration. Due to the lack of basic research in brain disorders most of the major pharmaceutical companies are abandoning drug discovery programs into treatments for these disorders. The social and healthcare costs of these diseases are enormous and therefore any advances in our understanding of the underlying mechanisms will impact on the treatment of these diseases and the development of disease specific drugs. This will greatly influence and benefit the health and quality of life of patients and their immediate social environment. This can create economic benefits by saving health care costs and give a boost to the UK-based pharmaceutical industry.
3. The General Public will benefit from this research, because it will further our understanding of the causes of disease and will demonstrate the enormous benefits of basic research to the wider society.