Pseudocoloured localisation images demonstrating the diversity in spatial organisation between cells expressing (a) TCR, (b) 1st generation CAR or (c) 2nd generation chimeric antigen receptors.
Image credit: Matt Graus and Kristen Feher
“T cell receptor signalling machinery is very sensitive to spatial segregation – one molecule shifting four to seven nanometres can either start or stop T cell receptor signalling.”
More than 30 years after the discovery of the T cell receptor, we still do not know how signalling begins. It is important that we work out how antigen binding initiates intracellular signalling because these signals shape the resulting immune response. With new single-molecule imaging approaches, we aim to map receptor signalling at the molecular level while retaining the spatial organisation of intact cells. We are developing new instruments, new analysis methods and new molecular biology tools in an interdisciplinary research program that spans physics, chemistry, mathematics and biology.
PROF. KATHARINA (KAT) GAUS
AT A GLANCE
Super-resolution fluorescence microscopy promises the opportunity to place single molecules and multi-molecular complexes in the cellular context and in turn understand how molecular organisation of cells leads to cellular outcome. A key question in cellular immunology is how antigen recognition leads to intracellular signalling, on which T cell fate decisions are based. The T cell signalling field is stymied as we do not understand how engagement of the T cell receptor (TCR) on the extracellular side initiates phosphorylation of the constitutively associated CD3 dimers on the intracellular side. Further, it is not known how diverse signalling outcomes are encoded by a common TCR-CD3 signalling transduction process. We hypothesise that the spatial organisations are key to signal initiation, integration of signals from multiple receptors, and the regulation of a highly plastic signalling network.
PUBLICATION HIGHLIGHTS
Coelho S, Baek J, Graus MS, Halstead JM, Nicovich PR, Feher K, Gandhi H, Gooding JJ, Gaus K. (2020) Ultraprecise single molecule localization microscopy enables in situ distance measurements in intact cells. Sci Advances. 17;6(16), eaay8271. doi: 10.1126/sciadv.aay8271.
Hilzenrat G, Pandizic E, Yang Z, Nieves DJ, Goyette J, Rossy J, Ma Y, Gaus K. (2020) Conformational states control Lck switching between free and confined diffusion modes in T cells. Biophys J. 118, 1489-1501. doi: 10.1016/j.bpj.2020.01.041.
COMPLETED ACTIVITY
- Identified subunits and subunit arrangements of protein complexes in intact cells, exploiting the ultraprecision of our new single-molecule localisation microscope (SMLM) called Feedback SMLM.
- Linked protein localisation and spatial organisations to function, distinguishing signalling from non-signalling molecules and determined the environmental factors.
- Combined biochemical assays with single-molecule imaging to establish a conceptual and theoretical framework of signal integration at the receptor level.
- Mapped signalling networks with novel protein probes and new statistical analysis for single-molecule data that reveals the information flow in intracellular signalling networks.
- Disseminated microscopy hardware and software. To make a lasting impact on the scientific community and connect with end-users, we are not just developing novel microscope hardware and software but also developing avenues to cost efficiently replicate and disseminate our approaches. For example, we are exploring how 3D printing could aid in the prototyping of hardware and collaborating with MASSIVE data processing facility to develop approaches for handling and processing large imaging-based data.