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Characterisation of the Formin Protein FHOD1 in Striated Muscle

Student thesis: Doctoral ThesisDoctor of Philosophy

Formin homology 2 domain containing protein 1 (FHOD1) is a diaphanous related formin of the FHOD subclass. In a similar manner to other formins, FHOD1 has primarily been found to regulate the polymerisation of actin-based structures in cells. In muscle, actin is a major constituent of both skeletal and cardiac myocytes, with the thin filament system of sarcomeres and the cytoskeleton being partly composed of actin. FHOD proteins have previously been highlighted as important regulators of muscle cell biology since FHOD3, a close relative of FHOD1 was shown to be essential for myofibrillar maintenance. There is little known about the regulation of actin-based structures in muscle cells. We therefore aimed to characterise FHOD1 and probe into its involvement in myofibrillar and cytoskeletal regulation. In this study of FHOD1 we addressed various aspects of the protein including its expression pattern and localisation, function, regulation, and novel interacting partners. Insight into the expression pattern of FHOD1 was gained by examining relative protein levels in different tissues, including both healthy and diseased heart. Subcellular localisation was addressed in a number of fluorescence microscopy experiments through antibody localisation studies in muscle tissue and cultured cells and through transient transfection of GFP tagged constructs. Expression of GFP tagged fragments and mutants helped to delineate the functional distribution of the FHOD1 molecule in cells. The function of the protein was further probed via molecular knockdown by RNAi and by looking at the capacity of FHOD1 to polymerise actin in cells. The role of formins in the heart was more broadly addressed by drug inhibition of their actin polymerising capacity. Previous studies have suggested that the Rho family of small GTPases as well as the Src kinases regulate FHOD1. Involvement of the GTPases and Src was shown through biochemical experiments. Finally, a number of Yeast2Hybrid assays were performed using different domains of FHOD1 to screen for novel binding partners. Our findings would suggest that FHOD1 is a crucial regulator of the myofibrillar apparatus and the cytoskeleton at the level of actin in striated muscle.
Original languageEnglish
Awarding Institution
Award date2015


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