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The cell membrane as a communication interface – use of minimal systems to study the principles of information transmission by anomalous diffusion

Primary Supervisor: Dr Darius Koester, Warwick Medical School

Secondary supervisor: Davide Calebiro, University of Birmingham

PhD project title: The cell membrane as a communication interface – use of minimal systems to study the principles of information transmission by anomalous diffusion

University of Registration: University of Warwick

Project outline:

The 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. All of this processing is reliant on a complex network of molecular interactions which are ultimately determined by diffusion.

The plasma membrane system consists of a lipid bilayer sandwiched between two dense, fibrous meshworks – the extracellular matrix/glycocalyx and cortical actin on the intracellular side. The bilayer itself is a complex diffusive environment consisting of viscous lipid domains, transient lipid trapping, dense protein-crowding, dynamic semi-permeable barriers and curvature. Communication, that is, signal transduction, from the extracellular space to the cytosol must therefore traverse this membrane system. Since transduction is also ultimately reliant on interactions governed by diffusion, the diffusive properties generated in this complex structure are important to understand.

We do not know how the composition and structure of the different interfacial elements create complex diffusive properties, nor do we know how the different modes of anomalous diffusion that are created influence the rates of molecular interactions. We also do not understand how the interactions at different stages of transduction generate the ultimate functions of signal processing, such as digitisation, integration and amplification.

We will focus the study 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 Here, we will make a first attempt to recapitulate the complex diffusive behaviour observed at the plasma membrane system. We will initially form planar supported lipid bilayers with integrated and associated proteins of interest, starting with membrane attached model proteins and then moving to purified GPCRs and G proteins. We will vary the lipid composition from simple to complex compositions resembling closer the plasma membrane. As a second step, we will implement passive and active actin networks using purified actin, actin nucleators and regulators, myosin II and ATP, allowing us to control the average network mesh size. We will attempt to tune these conditions to reflect cellular conditions and will compare the membrane protein mobility with our cellular observations. The composite actin-membrane system will allow us to determine what aspects of anomalous diffusion are retained on simplification of the biophysical system.

Techniques and methodologies the student will learn:

  • Total internal reflection fluorescence (TIRF), confocal and super-resolution microscopy
  • Quantitative image analysis using ImageJ, MATLAB, etc
  • in vitro reconstitution of SLBs, actin filament polymerization and handling of other purified proteins.
  • GUV formation and manipulation
  • Biochemistry/ molecular biology techniques

References:

  1. Sungkaworn T, Jobin ML, Burnecki K, Weron A, Lohse MJ, Calebiro D. Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature. 2017 Oct 26;550(7677):543-547
  2. Ditlev JA, Vega AR, Köster DV, Su X, Tani T, Lakoduk AM, Vale RD, Mayor S, Jaqaman K, Rosen MK. 2019. A composition-dependent molecular clutch between T cell signaling condensates and actin. Elife 8:e42695.
  3. 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.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Systems Biology

    Techniques that will be undertaken during the project:

    • Biochemistry and molecular biology techniques
    • Advanced microscopy (single-molecule, super-resolution)
    • Advanced image analysis
    • Protein purification and in vitro reconstitution of biological membranes

    Contact: Dr Darius Koester, University of Warwick