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Title: Arrangement and structure of α-actinins in striated muscle
Author: Rogers, Brendan James
ISNI:       0000 0004 7658 5228
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2018
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The smallest contractile unit within striated muscle cells are called sarcomeres. The boundary regions between sarcomeres are called Z-discs, which contain over 30 different proteins, organised within a narrow ~100 nm wide structure. Standard fluorescence microscopy approaches do not reveal the arrangement of Z-disc proteins, as the width of the Z-disc is below the resolution limit (~250 nm). The arrangement of the actin filaments and the cross-linking proteins α-actinin in the Z-discs are well characterised by electron microscopy (EM) studies, however other Z-disc proteins are not. With the development of super-resolution fluorescence microscopy techniques, it is now possible to obtain Z-disc protein localisation information. Here, dSTORM (direct Stochastic Optical Reconstruction Microscopy) was used, to investigate the arrangement of α-actinins in Z-discs of cardiomyocytes, and then the arrangement of the N-terminal ends of the giant protein titin in the Z-discs. Affimers were generated to bind α-actinin 2 and the N-terminal titin domains (Z1/Z2), to use as binders in dSTORM. Affimers are small (~12 kDa) non-antibody binding proteins, about 1/10th the size of antibodies, that can be selected to bind to a specific protein. The localisation data of dSTORM using the Affimer binders showed the same regular arrangement of α-actinins observed in EM studies. The use of dSTORM with Affimers also suggests the titin Z1/Z2 domains do not only localise at the edges of the Z-discs but arranged throughout the Z-disc with regular spacing (~25 nm) in the transverse plane of the Z-discs. Also, three mutations located in the actin binding domain of α-actinin 2 associated to hypertrophic cardiomyocytes (G111V, A119T and M228T) were characterised by in vitro co-sedimentation assays with actin. The mutants G111V and A119T did not show a significant difference in binding affinity to actin compared to the wild-type. The co-sedimentation assays did however suggest the mutation M228T significantly increases the binding affinity of α-actinin 2.
Supervisor: Peckham, Michelle Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available