Determination of laminin LN domain roles in tissue function, using Pierson syndrome and the lens capsule as a model.

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Basement membranes (BM) are specialised extracellular matrix structures, critical for tissue integrity. Moreover, BMs regulate key tissue behaviour through three distinct mechanisms: 1) By directly modulating signalling cascades, 2) Through sequestration followed controlled release of growth factors and 3) Through presenting context specific biomechanical cues to attached cells. Each of these properties has been shown to be dependent on BM composition, however, how the 3D organisation and macromolecular assembly status of those components determine tissue behaviour is less understood and has not been rigorously investigated. The laminins are a major structural component of all BMs. Each laminin is a heterotrimer capable of interacting with cell surface receptors (integrins, dystroglycan, syndecan) and other matrix proteins including nidogens and perlecan. Additionally, laminins assemble into networks, a feature controlled by sequences within their LN domains. Missense mutations in this domain are pathogenic and when they occur in LAMB2 they lead to Pierson Syndrome.The aim of this studentship is to determine the mechanisms through which LN domain mutations impact tissue function. Our focus will be Pierson syndrome, where patients present with lens abnormalities including abnormal shape and cataract formation. The lens capsule is a specialised BM that is one of the thickest human BMs, it that can be readily isolated and support the in vitro culture of cells. Moreover, the lens capsule contains a limited subset of laminins making it a tractable system for asking the focused questions proposed hereThis studentship will test the hypothesis that pathogenic mutations will influence the BM function in a context specific manner. The student will therefore characterise the effect of laminin mutants on:1) Laminin Signalling. Analysis of activity level of signalling pathways (candidate and hypothesis independent), comparative analysis of location, expression, interaction with mutants, and activity of laminin receptors.2) Growth factor sequestration by LAMB2. Immunogold EM quantification of perlecan/FGF2 abundance in BMs, immunoprecipitation for perlecan/nidogens, and functional analysis through resistance to staurosporine/etoposide induced apoptosis. 3) Structural/biomechanical properties of laminin networks. Laminin network morphology assessed by electron microscopy, biomechanical properties by atomic force microscopy and tensile strength analysis. To do so we will use CRISPR-Cas9 genome editing technology to introduce Pierson syndrome mutations into human lens epithelial cells (FHL124). We will then combine these cells, with isolated bovine lens capsules that have been treated to remove existing LMs to generate 3D physiologic ally relevant systems in which each of these potential BM functions can be assessed.