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Dr Andrew Shevchuk and his team from Imperial College London will investigate how mutations associated with some types of centronuclear myopathy and Charcot-Marie-Tooth disease (CMT) affect an essential cell process called endocytosis that allows cells to take up nutrients and replenish their cell membranes. Findings from this study will increase our understanding of the role of endocytosis in these conditions and could highlight potential therapeutic targets for the development of future therapies.
This project is co-funded by Muscular Dystrophy UK and CMT-UK – a collaborative partnership established to accelerate progress in the search for treatments and eventually cures for Charcot Marie Tooth disease.
Dr Shevchuk and his team have established a method that allows them to study the effects of mutations in the dynamin 2 gene that are linked to centronuclear myopathy (CNM) and Charcot-Marie-Tooth disease (CMT)on endocytosis. In endocytosis the membrane curves in to the cell and is pinched off to form very small (approximately one ten thousandth of a millimetre) ball shaped structures called vesicles, with the cargo inside. The team are currently investigating two types of endocytosis: clathrin-mediated and caveolin-dependent. They are named for the key proteins stabilizing the shape of vesicles and take up different types of cargo. Both require dynamin 2 to function.
Along with important laboratory techniques the PhD student has learnt how to operate state of the art correlative scanning ion conductance microscope and fluorescence confocal microscope (SICM-FCM) setup. The student uses this to look at how single endocytic vesicles form in skin cells from people with centronuclear myopathy and CMT. The researchers found that mutations in different parts of dynamin 2 have different damaging effects on how clathrin-coated vesicles form. Some of the team’s findings are different from results from other researchers. Dr Shevchuk and his student are currently using more techniques to investigate in greater detail how the mutations in dynamin 2 affect endocytosis.
This year the student has also learnt how to follow the formation of individual caveoli vesicles using correlative SICM-FCM. The researchers have also used SICM techniques to measure the stiffness of skin cells because this may be regulated partly by caveolin and clathrin.
Next the team will continue their in-depth analysis of clathrin- dependent endocytosis mechanisms in cells with dynamin 2 mutations. The researchers will look to see if the caveolin-dependent vesicles are affected in skin cells with the dynamin 2 mutations with SICM-FCM and if the stiffness of the cells with the dynamin 2 mutations is different from healthy cells.
Some types of centronuclear myopathy and Charcot-Marie-Tooth disease (CMT) can be caused by mutations in a gene that carries the genetic blueprint for a protein called dynamin 2. This protein plays a role in an essential cellular process called endocytosis. Cells use this process to collect nutrients and messenger molecules from their surroundings and to maintain the structure of the membrane that encloses them.
Until recently, few lab techniques were capable of measuring endocytosis in living cells and this has hampered understanding of the process and what happens when things go wrong with it. Dr Shevchuk and his team have developed a new technique called scanning ion conductance microscopy (or SICM) which allows them to observe endocytosis in living cells.
In this project, the researchers will use this technique to learn more about the effects that mutations in the dynamin 2 gene have on endocytosis. They will study endocytosis at the nanometre level (one millionth of a millimetre) in skin cells from people with centronuclear myopathy or CMT and compare them to cells from unaffected individuals. This will indicate precisely how the mutations disrupt endocytosis and at what stage of endocytosis the disruption occurs. This could identify potential targets for the development of future therapeutic approaches.
This project will reveal the changes in endocytosis that are caused by mutations in the dynamin 2 gene – responsible for some types of centronuclear myopathy and CMT. This will increase our understanding of the molecular mechanisms underlying these conditions and will be useful for future drug development work – it may help identify new therapeutics targets for future interventions.