Dynamics and oligomerisation of ABCG2 investigated using various fluorescence techniques.
PhD thesis, University of Nottingham.
The human ABCG2 (second member of ABC transporter G-subfamily) is an important ATP-dependent exporter in the body with broad substrate specificity including xenobiotics (e.g. anticancer agents) and endogenous compounds (e.g. sterols and lipids). ABCG2 was first discovered in a multidrug resistant breast cancer cell line and it is suggested to cause resistance to chemotherapy in certain cancers such as acute myeloid leukaemia and small cell lung cancer. Physiologically, ABCG2 is found in the protective sanctuary sites of the body, for instance the gut and blood-brain-barrier, affecting pharmacokinetics and treatment efficacies of small molecule drugs. Structurally, the polypeptide chain of ABCG2 contains a single nucleotide binding domain and a single transmembrane domain, which is half the number of domains required for a fully functional ABC transporter. Although many have suggested that ABCG2 function as dimer or higher order oligomer, studies so far have been unable to convincingly address the oligomeric state of ABCG2.
We aim to bridge this knowledge gap by resolving the oligomerisation of ABCG2 using fluorescence techniques in mammalian cells. The expression and function of fluorescent proteins tagged ABCG2 were verified using confocal imaging and fluorescence accumulation assays, prior to the fluorescence studies. As the membrane dynamics of ABCG2 are unknown, we first measured the diffusion of ABCG2 in live HEK293T cells using fluorescence recovery after photobleaching (FRAP) microscopy, in comparison to membrane localised fluorescent proteins and a full length (i.e 4 domain) ABC transporter (ABCC4). We also demonstrated oligomerisation of ABCG2 by measuring a specific increase in fluorescence resonance energy transfer (FRET) efficiency between CFP- and YFP-tagged ABCG2 expressed in live HEK293T cells in comparison to non-specific control interactions, including with the adenosine A3 receptor.
Subsequently, we employed high resolution and single particle fluorescence techniques to resolve the oligomeric organisation of ABCG2. First, fluorescence fluctuations of tagged ABCG2 within a confocal volume, positioned on the upper plasma membrane, were measured using fluorescence correlation spectroscopy (FCS) at “single molecule” resolution. Photon counting histogram (PCH) analysis of the FCS measurements was performed to determine the molecular brightness of the fluorescent species detected within the confocal volume. Using CD86 and CD28 as monomer and oligomer controls respectively, PCH analysis demonstrated higher order oligomer formation of ABCG2, with increased brightness (up to 4-fold) observed for both ABCG2 and CD28, compared to CD86. For validation of the oligomeric organisation of ABCG2, we acquired a series of single particle photobleaching images of cells expressing fluorescent protein tagged ABCG2 using total internal reflection fluorescence (TIRF) microscopy at the lower plasa membrane, and employed a step detection algorithm to identify the number of photobleaching steps within the distinguished fluorescent spots. Statistical modelling of the photobleaching step frequency histogram provided credible evidence of tetrameric organisation of ABCG2 in the plasma membrane. The findings and methodology presented in this study have provided further insights into the membrane dynamics and oligomerisation of ABCG2. This could lead to future studies to explore new pharmacological avenues that target the oligomerisation interfaces of ABCG2.
Thesis (University of Nottingham only)
||Q Science > QP Physiology > QP501 Animal biochemistry
QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
||UK Campuses > Faculty of Medicine and Health Sciences > School of Biomedical Sciences
||19 Oct 2015 10:04
||14 Sep 2016 08:54
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