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Investigating cell-to-cell communication via tetraspanin-containing extracellular vesicles

Primary Supervisor: Dr Mike Tomlinson, School of Biosciences

Secondary supervisor: Dr Paul Harrison

PhD project title:Investigating cell-to-cell communication via tetraspanin-containing extracellular vesicles

University of Registration: University of Birmingham

Project outline:

Background

The transport of cargo between cells via extracellular vesicles is an emerging mechanism by which cells communicate with each other. Such cargo includes proteins and RNA that modify function in acceptor cells, affecting immune responses, cancer and other processes in health and disease. There are two major types of extracellular vesicles: 100-1000 nm vesicles that bud from the plasma membrane and are termed microvesicles, ectosomes or microparticles; and 50-150 nm vesicles that originate from the endocytic trafficking pathway, termed exosomes. The latter derive from intraluminal vesicles within multivesicular bodies, which are part of the late endocytic trafficking pathway, and are released upon fusion of multivesicular bodies with the plasma membrane. Despite these recent advances in the extracellular vesicle field, the mechanisms of extracellular vesicle formation and their functions in cell-to-cell communication remain poorly understood.

A striking feature of extracellular vesicles, particularly exosomes, is their relatively high expression of tetraspanin membrane proteins. Indeed, tetraspanin antibodies are used in exosome purification and quantitation techniques. The tetraspanins are a superfamily of 33 proteins in humans, with a four-transmembrane structure and a characteristic cone shape, which may facilitate the curvature in membranes needed for extracellular vesicle formation. However, the role of tetraspanins in extracellular vesicle formation and function is not clear. Tetraspanins function by interacting with specific membrane proteins, termed partner proteins, and regulating their trafficking to the cell surface, lateral diffusion and clustering. A partner protein of interest to our group is the ADAM10 molecular scissor. ADAM10 is a metalloprotease expressed by all cells and has critical roles in health and disease by cleaving the extracellular regions from it substrates, termed ectodomain shedding. ADAM10 substrates include the cell fate regulator Notch, amyloid precursor protein that is important in Alzheimer’s disease, cadherin adhesion molecules and growth factors. We have identified six tetraspanins that regulate ADAM10, and have generated data to support a ‘six scissor’ hypothesis, whereby ADAM10 substrate specificity is dictated by which of the six tetraspanins it is associated with. We propose that extracellular vesicles provide a means for one cell type to transfer a tetraspanin/ADAM10 scissor to another cell type that would not normally express that tetraspanin, to modulate shedding and function in the acceptor cell. This idea has yet to be investigated but is a focus of this project.

Objectives

  1. To investigate whether specific tetraspanins preferentially localise into extracellular vesicles.
  2. To determine whether tetraspanins are important for extracellular vesicle formation.
  3. To discover whether extracellular vesicle-mediated transfer of tetraspanins and associated membrane proteins can modulate function in the acceptor cell.

Methodology

Culture of human cell lines; transfection to establish tetraspanin over-expressing and CRISPR/Cas9-knockout cells.

Western blotting using the Odyssey quantitative infrared imaging system and flow cytometry to quantitate tetraspanin expression.

Quantitation and characterisation of extracellular vesicles using ExoView technology.

Advanced fluorescence microscopy (e.g. super-resolution and lattice light sheet): investigating tetraspanin subcellular localisation; imaging extracellular vesicle release by donor cells and uptake by acceptor cells.

ADAM10 substrate shedding and cell function assays.

References:

  1. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Mathieu M, Martin-Jaular L, Lavieu G, Théry C. Nat Cell Biol 2019 21:9-17.
  2. Scissor sisters: regulation of ADAM10 by the TspanC8 tetraspanins. Matthews AL, Szyroka J, Collier R, Noy PJ, Tomlinson MG. Biochem Soc Trans 2017 45:719-730.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology

    Techniques that will be undertaken during the project:

    Cell culture, transfection, CRISPR/Cas9 gene editing, Odyssey western blotting, ExoView extracellular vesicle characterisation, advanced fluorescence microscopy (super-resolution and lattice light sheet), cell surface protein shedding and in vitro cell function assays.

    Contact: Dr Mike Tomlinson, University of Birmingham