Single-molecule analysis of tropomyosin to filamentous actin interactions

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Copyright: Obeidy, Peyman
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Abstract
The actin cytoskeleton plays a fundamental role in many biological processes including cell proliferation, cell shape, cell migration and intracellular transport. This enormous functional range is associated with differences in filament composition due to interaction with actin-binding proteins. Tropomyosins are key players in the dynamic regulation of the functions of F-actin. However, the molecular mechanisms underlying the assembly of tropomyosin strands on an actin filament, including the competition between different tropomyosin isoforms and the role of the N- and C-terminus overlap regions, are largely unknown. The main roadblock for addressing these questions is a lack of techniques for direct visualisation of tropomyosin to actin filament interactions, and complementary image processing tools. For this study, a sensitive single-molecule fluorescence imaging assay was obtained by combining TIRF microscopy and microfluidics. Cytoskeletal and skeletal tropomyosin isoforms were fluorescently labelled using maleimide chemistry, and the functionality of the labelled proteins were validated using standard biochemical assays. Actin filaments were exposed to labelled tropomyosin isoforms and captured on the surface of a microfluidic channel for visualisation by TIRF microscopy. Snapshots of the early stages of the assembly process of tropomyosin dimers on F-actin were quantified using automated image analysis. Our observations reveal nucleation of short stretches of tropomyosin polymers at multiple locations along the actin filaments. Our data suggest that affinity does not play an important role in the sorting of muscle and cytoskeletal tropomyosin isoforms to their corresponding actin isoforms. At the level of individual filaments we observed some degree of segregation of tropomyosin isoforms with different C-termini to distinct clusters on F-actin. The platform developed for this study represents an enabling technology for real-time observations of tropomyosin to actin filament assembly and disassembly pathways.
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Author(s)
Obeidy, Peyman
Supervisor(s)
Boecking, Till
Gunning, Peter
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Publication Year
2015
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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