The basement membrane (BM) is an essential extracellular matrix (ECM) composed of a polymeric network of components underlying all epithelia. BM structure and mechanical properties are altered in several pathologies. Additionally, inherited mutations in Collagen IV (Col4), a major BM component required for BM stability, lead to a severe multi-system disorder called Gould syndrome. Disease severity is dependent on the position of the Col4 mutation leading to varying degrees of BM disruption through mechanisms that are currently unclear.
Here we will use Drosophila to understand the physiological consequences of Collagen IV (Col4) mutations associated with disease. By exploiting our unique capacity to generate precise Col4 mutations and analyse structural changes to the Col4 polymer network within tissues, we will unveil the precise effects of different Col4 mutations on BM architecture. This will allow us to understand how distinct Col4 alterations drive disease phenotypes.
The prospective PhD candidate will gain experience with molecular biology; genetics; super-resolution microscopy; and cryo-electron microscopy (EM), with skills developed to being a fully independent operator of a cryo-focused ion beam scanning EM, cryo-correlative light to EM imaging system, and cryo-transmission EM. This will be supported by collaborations with instrument developers. The student will also gain experience with image processing and the application of AI in image collection.
The aim of the project is to use high resolution imaging techniques to first characterise the ultrastructure of the BM during Drosophila development and adult stages, and subsequently analyse structural changes in Col4 mutations that model human disease. The student will also gain exposure to human Col4 mutations and disease phenotypes through collaborative interactions with scientists studying Gould syndrome and the prevalence of Col4 mutations in the UK Biobank with the goal of extrapolating our understanding to the human disease.
PhD Objectives:
Year 1: Characterise normal Col4 structure in developing and adult tissues using super-resolution imaging techniques by taking advantage of our capacity to fluorescently tag the Col4 protein.
Years 1-2: Develop a cryo-EM pipeline to understand the ultrastructure of the normal Col4 network. Potentially develop correlative approaches to connect super-resolution and EM imaging.
Years 3-4: Analyse the effects of dominant Col4 mutations on BM architecture in vivo and correlate structural defects with the degree of mutant lethality.
Rotation Project:
The PhD candidate with gain experience with genetics and super-resolution imaging of normal and mutant Col4 to gain a preliminary understanding of BM alterations associated with disease-associated mutations.
