Protein segregation and conformation in antigen-receptor triggering: a quantitative fluorescence microscopy study

Antigen receptors play a central role in determining whether an immune response is required. The remarkable diversity of their extracellular domains allows them to detect unfamiliar pathogens. In response, information is conveyed across the plasma membrane, resulting in the phosphorylation of cytoplasmic domains. This process, known as triggering, has been the subject of much controversy. Antigen-independent triggering has been demonstrated for the T-cell receptor, challenging conventional views of signal transduction. The kinetic-segregation model proposes that size-dependent exclusion of phosphatases increases net receptor phosphorylation. Rather than initiating downstream signalling autonomously, ligand binding serves to hold the receptor within phosphatase-depleted regions of the membrane. The strength of this model is that receptor-ligand interactions are considered in the broader context of their physical environment. This thesis asks how intermembrane distance affects antigen-receptor triggering. Total internal reflection fluorescence microscopy is used to establish that phosphatase exclusion decreases when intermembrane distance increases. This inverse relationship, demonstrated for both B cells and T cells, is consistent with the kinetic-segregation model. However, in T cells, increases in intermembrane distance are not found to affect downstream signalling. The experiments described here thus fail to establish a relationship between phosphatase segregation and triggering. Part of this thesis addresses an unanticipated problem with the experimental system, namely that lymphocytes are triggered by nickel-chelating lipids in supported lipid bilayers. An effective and easily implemented solution is identified, which can be used in future work with this popular model surface. A short section focusing on the kinase Lck is also included. The feasibility of using Förster resonance energy transfer to identify spatiotemporal changes in Lck conformation is explored. Understanding antigen-receptor triggering is crucial, as failure to discriminate between self and non-self antigens can have life-threatening consequences. Here, live-cell imaging is used to examine kinase and phosphatase behaviour, in an attempt to shed some light on how decisions are made.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Humphrey, Jane

Advisor dc:contributor.advisor

• Klenerman, David

• antigen-receptor triggering
• kinetic-segregation model

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/002a953a-9943-4b29-bd35-7d028567d7b6/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

Author Identifier

0000-0002-6825-1426
0000-0001-7116-6954

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/323308