Principal Supervisor: Dr Darius Vasco Köster, Warwick Medical School
Co-supervisor: Matthew Turner
PhD project title: Reconstitution of the cell plasma membrane-actin cortex interface
University of Registration: University of Warwick
Cells are in constant exchange of information and material with their surroundings, mediated via the cell surface, i.e. the plasma membrane and the underlying cytoskeleton. The plasma membrane is composed of thousands of different lipid species and proteins and transient, local organisation is thought to be crucial for many processes such as the uptake or release of cargo, signalling or cell adhesion. While it is well established that the plasma membrane and the underlying cytoskeleton are closely intertwined, only recently more and more experimental and theoretical evidence is underlying the important and imminent role of the actin network dynamics on local membrane organisation and many aspects on the molecular, physical and cellular level of this interaction are not yet understood.
The aim of the proposed project is to reveal fundamental, physical mechanisms underlying the versatile and adaptive nature of the cell surface using a bottom-up approach based on supported lipid bilayers and purified proteins. In previous work, we established a basic acto-myosin network linked to supported lipid bilayers of only one lipid component and studied how acto-myosin activity can drive clustering and the dynamics of lipid anchored proteins (1, 2). The key aim of this project is to study the impact of acto-myosin activity on lipid organization and phase separation by implementing tested and newly developed design strategies to connect specific subsets of lipids to the acto-myosin network. Preliminary work in the lab (Fig. 1) and elsewhere (3)indicates that lipid pinning by actin filaments induces local formation of lipid domains. But how this process is affected by acto-myosin activity, the strength of actin-lipid anchoring and the lipid composition, and whether this effect could be the basis of a feed-back mechanism that is important in local signalling or activation of actin polymerization in cells, are open questions that we try to answer with this project.
The first steps of the project will involve the characterization of ‘simple’ three component lipid systems and comparing their phase separation and lipid organisation at different temperatures in the absence or presence of static actin networks using fluorescent lipid probes and total internal reflection fluorescence (TIRF) or confocal microscopy. Introducing myosin II motor proteins and ATP will lead to active remodelling or contracting acto-myosin networks that will change the dynamics of lipid organisation. An additional parameter will be the anchoring strength between lipids and actin, which will be controlled using actin-membrane linking proteins of differing binding affinity. Findings of this research will later be tested in experiments on live cells and/or by using more physiologically relevant membrane compositions.
In collaboration with the group of Matthew Turner, we will test and develop novel theoretical models describing lipid organisation in active membranes. We will also develop analytical methods to derive quantitative measures of lipid flows and acto-myosin network dynamics from the acquired fluorescence images using imageJ and Matlab.
This project will provide major advances in our understanding of the dynamic regulation of cell membrane organisation and molecular details of processes involved in cell signalling.
- Köster DV, et al. (2016) Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer. Proc Natl Acad Sci113(12):E1645–E1654.
- Ditlev JA, et al. (2018) A Composition-Dependent Molecular Clutch Between T Cell Signaling Clusters and Actin. bioRxiv. doi:10.1101/316414.
- Honigmann A, et al. (2014) A lipid bound actin meshwork organizes liquid phase separation in model membranes. Elife3:e01671.
BBSRC Strategic Research Priority: Molecules, Cells and SystemsTechniques that will be undertaken during the project:
- Molecular/synthetic biology: plasmid design and construction, protein expression and purification (in cells and bacteria)
- Microscopy: Fluorescence light microscopy (confocal, TIRF, super resolution)
- Biomimetic systems: lipid handling, supported lipid bilayer formation, substrate modifications (patterning, coating, stiffness modulation)
- Data analysis: Quantitative image analysis, programming of analysis routines (e.g. in Matlab) (optional), Computational modelling (optional).