Tropomyosin isoforms show unexpected differential effects on actin polymerization
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AbstractTropomyosin is a rod-like coiled-coil protein that forms a continuous filament that is weakly associated, but firmly-attached to the surface of the actin filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only one or two different tropomyosin isoforms which are known to be essential and perform roles in regulating the actin cytoskeleton. However higher eukaryotes have larger numbers of tropomyosins, the number of which appear linked to organismal complexity. Mammals have 4 genes producing over 40 different isoforms by alternative splicing. In higher organisms tropomyosin is best known and characterized in the regulation of striated muscle contraction. The role of tropomyosin outside of muscle is less well understood. It is generally thought to have a regulatory role in controlling interactions of actin-binding proteins and in providing additional stability to actin-filaments. In the latter case has been considered that tropomyosin binds to actin-filaments some time after their formation, both making them mechanically stiffer and protecting them from breakdown. We have produced a range of recombinant tropomyosins from all four mammalian genes and characterized their actin-binding affinities in a cosedimentation assay. We have then used them to systematically study the effects of different isoforms of tropomyosin on actin polymerization for the first time. We have monitored actin polymerization by the well-characterised change in fluorescence of a pyrene-label attached to actin. Actin polymerisation is monitored by measuring the significant fluorescence enhancement on polymerization. Our results characterize the actin-affinities of some of the TPM3 and TPM4 isoforms for the first time, These are in the same general range as mammalian isoforms previously characterized by our group and others. We demonstrate differential effects of the different isoforms on actin-polymerisation for the first time. The data unexpectedly show the most significant effects of the different isoforms appears to be in the early initiation / elongation stages of polymerizations. This is unexpected as tropomyosin is only considered to have significant affinity for actin filaments through itself forming a polymer along the surface of an actin filament. Different isoforms appear capable of both enhancing and inhibiting the early stages of polymerization, with examples of the shorter 6-actin spanning TPM1 gene isoforms showing a significant reduction in the lag-phase of early polymerization. These differential effects on different isoforms provides a new role for tropomyosin in not only stabilizing filaments, but also in helping catalyze their formation.
CitationMaytum R, Dudekula K (2017) 'Tropomyosin isoforms show unexpected differential effects on actin polymerization', Biophysical Soceity Annual Meeting - , American Chemical Society.
PublisherAmerican Chemical Society
TypeConference papers, meetings and proceedings
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