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Understanding by creating – minimal systems to reveal the underlying mechanisms governing the cell membrane shape and function

Principal Supervisor: Dr Darius KoesterLink opens in a new window

Co-supervisor: Davide Calebiro (U of Birmingham), Mark Wallace (King’s College London)

PhD project title: Understanding by creating – minimal systems to reveal the underlying mechanisms governing the cell membrane shape and function

University of Registration: University of Warwick


Project outline:

The cell plasma membrane is essential for communication between the cell and its environment. It is here that crucial information processing takes place – including steps of signal detection, integration, amplification and transmission. This processing relies on a complex network of molecular interactions and many diseases are linked to failures in this information processing.

The plasma membrane system consists of a lipid bilayer sandwiched between two dense, fibrous meshworks – the extracellular matrix and the actin cortex on the intracellular side. The lipid bilayer itself is a complex diffusive environment consisting of densely packed lipid domains, protein-crowding, dynamic semi-permeable barriers, and curvature. Any signal transmission from the outside towards the inside is passed on via membrane proteins and many key aspects of this process are not well understood.

Two projects are aimed to improve our understanding of the role of lipid membrane organisation and signal transmission.

  • GPCR signalling and anomalous diffusion (Davide Calebiro, U of Birmingham)

The Calebiro lab works on G-protein coupled receptors (GPCRs), the largest family of membrane receptors. GPCRs mediate the effects of several hormones and neurotransmitters and are the targets of more than 30% of all drugs on the market. Once activated by an extracellular agonist, GPCRs interact with membrane-bound G proteins that relay the signal to effectors, also located on the plasma membrane.

The aim of the project is to build a minimal in vitro system to recapitulate observed diffusional complexity of membrane proteins and study its impact on receptor-G protein interactions, chosen as a model of a fundamental protein-protein interaction involved in cell communication. Using supported lipid membranes, purified GPCRs and cytoskeletal proteins, we will make a first attempt to recapitulate the complex diffusive behaviour observed at the plasma membrane system by controlling every aspect of the system.

 

  • How the cytoskeleton controls the organisation of cell membranes (Mark Wallace, King’s College London)

Lipid organisation into domains is crucial for cell signalling, and the concept of liquid-liquid phase separation as such an organising principle for lipids and proteins has gained new attention. In the cell membrane, the interaction with the underlying, dynamic actin network is also important, and it is the interplay of both mechanisms, condensation into domains and active forces by the acto-myosin network that is essential for understanding membrane control.

Our previous work has shown that acto-myosin activity counters the formation of lipid domains. Based on this, we want to address the following intriguing questions fundamental to the understanding of cell signalling: How does lipid domain formation affect the organisation of actin networks, and how do mechanical forces of the acto-myosin network affect lipid domains.

This interdisciplinary work will establish a new framework for the understanding of cell membrane organisation and will be undertaken together with the Wallace laboratory who have pioneered the Droplet-Interface-Bilayers, a free-standing lipid membrane system, that is ideal for the study of dynamic lipid domains.

Both projects are exciting and will be based in the Koester laboratory that has expertise in the building and the study of acto-myosin networks on lipid membrane systems in combination with state-of-the-art fluorescence microscopy and quantitative image analysis. It is located in the Centre for Mechanochemical Cell Biology which offers an open lab space that makes interaction with other labs easy and offers a modern work environment.

 

References:

Köster DV, Husain K, Iljazi E, Bhat A, Bieling P, Mullins RD, Rao M, Mayor S. (2016) Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer. Proc Natl Acad Sci 113:E1645–E1654

Sungkaworn T, Jobin ML, Burnecki K, Weron A, Lohse MJ, Calebiro D. (2017) Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature. 550(7677):543-547

Leptihn, S., Castell, O. K., Cronin, B., Lee, E. H., Gross, L. C. M., Marshall, D. P., Thompson, J. R., Holden, M., & Wallace, M. I. (2013). Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nature Protocols, 8(6), 1048–1057.

BBSRC Strategic Research Priority: Understanding the rules of life Systems Biology.

 

Techniques that will be undertaken during the project:

  • in vitro reconstitution of supported lipid bilayers, actin filament polymerization and handling of other purified proteins.
  • Droplet interface bilayer formation
  • GUV formation and manipulation
  • biochemistry/ molecular biology techniques
  • total internal reflection fluorescence (TIRF), confocal and super-resolution microscopy
  • Quantitative image analysis using ImageJ, MATLAB, etc

Contact: Dr Darius KoesterLink opens in a new window