Using super resolution microscopy to investigate the role of actin in adenosine receptor organisation

Garlick, Evelyn (2023) Using super resolution microscopy to investigate the role of actin in adenosine receptor organisation. PhD thesis, University of Nottingham.

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Membrane receptors are key to how cells interact with other cells and their environment. G Protein-Coupled Receptors (GPCRs) are a major drug target, with approximately a third of all FDA approved drugs acting on a GPCR [1]. The organisation of GPCRs in the cell membrane can play a key role in determining signalling responses and associated pharmacological parameters. There is significant evidence that the cortical actin skeleton can contribute to this organisation via the picket fence model. The direct contribution of actin architecture and dynamics to organisation of specific receptors requires further study. Therefore, this thesis applies a range of super-resolution microscopy techniques to investigate the role of cortical actin in the organisation of the human adenosine-A2A (A2AR) and -A2B receptors (A2BR).

Using A549 cells transiently transfected with N-terminally SNAP-tagged receptor constructs, clustering analysis using dSTORM (direct stochastic optical reconstruction microscopy) indicates effects of actin disruption on A2AR clustering but not A2BR, while assessment of dynamic behaviour via single particle tracking (SPT) indicates differential effects on the motion patterns of each receptor. This was further supported by 3D-SIM (structured illumination microscopy) imaging of actin and receptors together. Assessment of actin using SRRF (super resolved radial fluctuations) processing showed a change in actin architecture after receptor stimulation. Workflows for imaging and analysing finer actin filaments using 3D-SIM expansion microscopy (ExM) were also developed, with incorporation of the A2R interacting protein α-actinin-1 serving both as investigation of a potential actin link and as a demonstration of two colour ExM. Initial experiments using SRRF processing indicated super-resolution imaging of actin was possible on a timescale which allowed concurrent single particle tracking of receptors, opening potential for correlated analysis.

These findings indicate a role for actin in mediating A2AR and A2BR membrane organisation, with potential for different regulatory contributions between receptors and across organisational scales.

[A thesis submitted to the University of Birmingham and the University of Nottingham for the dual degree of Doctor of Philosophy, August 2022.]

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Thomas, Steve
Briddon, Steve
Keywords: Cortical actin, Membrane receptors, Human adenosine receptors, Microscopy
Subjects: Q Science > QH Natural history. Biology > QH201 Microscopy
Q Science > QH Natural history. Biology > QH573 Cytology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 72356
Depositing User: Jacob, Mr Tim
Date Deposited: 29 Mar 2023 08:16
Last Modified: 29 Mar 2023 08:16

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