The role of tropomyosin in regulating actin filament dynamics in fission yeast

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences


Actin is an essential cytoskeletal protein which is conserved in all eukaryotic organisms examined to date. It is actin's ability to polymerise into dynamic filaments which allows a cell's growth and even movement to be rapidly affected by both intra- and extra-cellular demands. Upon cellular signalling the actin polymerises to form filamentous or F-actin which form both cables and lattice structures, known as patches in yeasts. These actin cables have been shown to have a role in a large number of cellular processes which include cell polarity; cytokinesis; cell growth and movement; providing cortical tension; endocytosis; and acting as 'pathways' along which molecular motors (myosins) can travel. Actin's functions are conserved within yeasts, and forms cables and patches which localise predominantly to regions of cell growth, facilitating the cells increase in size during interphase, or division during cytokinesis. The fission yeast, Schizosaccharomyces pombe, is cylindrical in shape, with growth occurring in a polarised manner at the cell pole. Actin is seen to localised predominantly to patch structures at these growing cell poles and to actin cables throughout the cytoplasm during interphase. During mitosis actin cables exist as a major component of the cytokinetic ring, which contracts in order for a cell to divide. This project make use of the cross-discipline approaches allowed by this experimentally tractable organism to determine the role actin filaments play in regulating and maintaining polarised cell growth. Using the versatile fission yeast model system allows us to not only examine actin filaments in both an in vivo and in vitro context, but allows us to make use of mutants to modulate filaments' dynamics as well as their ability to interact with motor proteins. Thus it will be possible to elucidate their function(s) within the cell.

Technical Summary

This project will address two main questions. The first is to determine the role actin filaments play in regulating and maintaining polarised cell growth in the fission yeast cell. The second is to determine whether tropomyosin plays a role in regulating myosin's interaction with actin and how it does this in a non-muscle cell. We will use real time live cell imaging to study actin filament dynamics in vivo, and examine the affect mutations within their component proteins (e.g. tropomyosin) has upon their dynamic properties, as well as establishing the affect the mutations have upon myosin function. We will go on to use a cross discipline approach to screen for novel tropomyosin mutants both in vivo (by identifying cells with abnormal actin filament function in the fission yeast cell) and in vitro (by identifying mutations which affect the protein's physical properties and interactions with actin). We will also explore the significance of why only a proportion of the tropomyosin in the fission yeast cell is constantly acetylated (80%) during the cell division cycle. This will be examined in detail using in vitro motility assays, as well as other biochemical techniques, and will examine how this post-translational modification affects tropomyosin's assocation with actin in a structural context using EM reconstruction techniques.


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East DA (2011) Regulation and function of the fission yeast myosins. in Journal of cell science

Description Actin filaments are dynamics structures that grow and shrink in response to the needs of the cell and are involved in a wide range of cellular processes. These filaments act as tracks for a series motor proteins, called myosin's. Different myosins undertake different tasks at different locations within the cell.
There were two major outcomes from this project.
Firstly, that a conserved actin associated molecule called Tropomyosin, plays a critical role in regulating the correct localisation and function of the different motor proteins in the cell.
Second, we discovered that a class of proteins, called formins, that "nucleate" actin filaments can determine which tropomyosin form associates with a specific actin filament.
Exploitation Route They have provided the basis for a variety of different studies in different cell types, and provide fundamental understanding of how different actin filaments are fine tuned at different cellular locations to facilitates distinct cellular activities.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Title Technology to allow amino-terminal acetylation of recombinant proteins in E. coli 
Description One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. We have generated a system that allows co-expression of the fission yeast NatB complex with the target protein in E.coli and allows to the expression and purification of functional N-terminally acetylated eukaryotic proteins. This technology allows significant savings in time and money over current techniques for generating amino-terminally acetylated recombinant polypeptides for both research and industrial applications, and is being widely used by the academic and biotechnology sectors. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Since its description in 2010, this reagent has already been sent out to more than 100 research labs worldwide (once an MTA document has been signed), and has already not only led to a series of publications, but has had a significant impact on a number of research fields.