Actin cytoskeleton-mediated force generation during T-cell activation investigated by advanced (super-resolution) microscopy

Supervisors: Dr Marco Fritzsche and Prof Christian Eggeling

Background
Over recent years, several examples have shown that the actin cytoskeleton plays an important role in actively reorganising molecules in the plasma membrane during cellular signalling. This notably holds for the cortical actin cytoskeleton of immune cells that is thought to be somehow involved in the coordination of their activation at all stages (1). Understanding the role of the actin cytoskeleton is now becoming one of the most contentious questions in immunology but progress has previously been limited, mainly due to the use of conventional-resolution microscopy, which inevitably misses essential details due to limited resolution. While these imaging techniques are currently probably the most valuable tools for directly investigating the living cell with minimal invasion, similar objects closer together than approximately 200 nm cannot be distinguished and details of the molecular organisation and dynamics of cytoskeleton-associated structures cannot be recovered directly. A remedy to this are recently developed more advanced microscopy techniques, such as super-resolution optical nanoscopy (2), actin-specific turnover measurements (3), the from us improved method of traction force microscopy (4) by using STED nanoscopy, as well as the simultaneous measurement of molecular turnover and forces by combining fluorescence spectroscopy and atomic force microscopy (AFM) (5).

Project
We aim to characterise the molecular reorganisation dynamics of the actin cytoskeleton that lead to active force generation during T-cell activation by the simultaneous read-out of the turnover dynamics of actin and key proteins involved in the activation process such as the T-cell receptor and forces applied by the T-cell. We expect these novel experiments to highlight, in thus far unprecedented detail, the role that the actin cytoskeleton plays in the activation of T-cells, and to create a critical framework for understanding immune responses.

Training Opportunities
This project will be based in the MRC Human Immunology Unit at the Weatherall Institute of Molecular Medicine, with access to state-of-the-art facilities. The project provides an opportunity for training in a broad range of different techniques, such as cell culture, molecular biology, and advanced microscopy, specifically including super-resolution optical microscopy and force measurement techniques such as STED or AFM. The disclosure of novel details of T-cell activation is an important line of basic immunological research that may translate into new approaches of modulating the immune response during infection.

Theme
Immunology, actin cytoskeleton, super-resolution microscopy, atomic force microscopy.

Publications

  1. A.A. Smoligovets, A.W. Smith, H.-J. Wu, R.S. Petit, J.T. Groves. Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells. J Cell Science (2012).
  2. C. Eggeling, K.I. Willig, S. J.Sahl, S.W. Hell. Lens-based fluorescence nanoscopy. Quart Rev Biophys (2015).
  3. M. Fritzsche, A. Lewalle, T. Duke, K. Kruse, G. Charras. Analysis of turnover dynamics of the submembranous actin cortex. Mol Biol Cell (2013).
  4. H. Colin-York, C. Eggeling, M. Fritzsche. Dissection of mechanical force in living cells by super-resolved traction force microscopy. Nat Protoc. (2017).
  5. E. Moeendarbary, A.R. Harris. Cell mechanics: principles, practises, and prospects. WIREs Systems Biology and Medicine.

For further information please contact Dr Marco Fritzsche