title: From single cell to single molecule: elucidation of the intracellular dynamics of the Hypoxia Inducible Factor (HIF)
Creators: name: Taylor, Sarah
Publisher: name: University of Liverpool Repository
Year: 2016

description: Hypoxia signalling pathway acts in all mammalian cells to try and avert damage occurring during periods of limited oxygen availability (hypoxia). The cellular response to a low oxygen environment is mediated by the transcription factor hypoxia-inducible factor (HIF), which activates the transcription of genes that aid cell survival and act to restore oxygen homeostasis. The heterodimeric transcription factor consists of a constitutively expressed beta subunit (HIF-1β) &amp; an alpha subunit (HIF 1α / HIF 2α). The alpha subunits are regulated in an oxygen-dependent manner. The main aim of this thesis was to elucidate if the spatial localisation and/or the temporal fluctuations of HIF-1α and HIF-2α are altered in response to hypoxia and participate to the regulation of HIF activity. The experimental strategy focused on studying HIF at several scales ranging from single cell to single molecule level. I first investigated the temporal and spatial dynamics of the HIF-α subunits in living cells using time-lapse imaging. This was part of a larger study, which was initiated before the start of this thesis. It was published in January 2014 in the Journal of Biological Chemistry. We demonstrated that HIF-α accumulates transiently in the nucleus in response to hypoxia and this was necessary to ensure cell survival. We further demonstrated using a combination of mathematical modelling and knock-down experiments that the negative feedback loop involving PHD2 has an essential role in these dynamics. Secondly, we observed that HIF-2α exhibits a non-homogenous sub-nuclear localisation, whereas HIF-1α is distributed homogenously within the nucleus. Since protein localisation is commonly linked to function we sought to elucidate the purpose of this heterogeneous distribution using a combination of live cell imaging, co-localisation and photo-bleaching experiments. Lastly, to avoid molecule averaging errors, we planned to employ single molecule tracking to elucidate sub-cellular dynamics of HIF-2α and gain further insight into the movement of HIF 2α between the speckles and the rest of the nucleus. Our aim was to image and monitor gold nanoparticles (GNPs) conjugated to HIF-2α in real time in living cells, using photo-thermal microscopy. In collaboration with Prof. D. G. Fernig and Dr. R. Levy’s research groups, we developed a protocol using the Halotag (an engineered enzyme that reacts with a substrate (Halo-ligand) to form a covalent bond) to label a protein of interest with gold nanoparticles. This was achieved by: immobilising the Halotag ligand on the surface of GNPs and producing purified recombinant Halotag-fusion protein. We have obtained positive results by using the fibroblast growth factor 2 as proof of principle. Due to difficulties in expression and purification of recombinant Halotag-HIF-2α single molecule tracking of HIF-2α has not been achieved yet, but this work is still on-going. Overall my results show that: (1) the temporal dynamics of HIF-1α elicited by hypoxia have a critical role in HIF activity and function, (2) the non-homogenous speckle localisation of HIF-2α is associated with a slow diffusion time within the nucleus, 7-10X (depending on condition) slower than the homogeneous HIF-1α. Finally, using single molecule measurements, we did not observe any significant difference in speckle organisation or HIF 2α mobility, so single molecular tracking will be required to provide a non-average quantitative measurement of HIF-2α movement between the speckles and inter-speckle space.