The Role of Meckel-Gruber Syndrome Proteins in Cell Migration

Nearly all cells in the human body form an organelle called a cilium, which acts as a cellular antenna. Cilia are important in cell morphogenesis, tissue function and embryonic development, and there are many inherited diseases caused by mutations in genes involved in cilium formation or function. However, we have little idea what the encoded proteins actually do in cells, or how mutations cause the disease. Consequently many of these diseases are untreatable. Our work focuses on a disease called Meckel-Gruber syndrome (MKS). Patients with MKS seldom survive longer than a few days due to the severity of the disease, which affects the brain, kidneys, liver and skeleton. We use genetics and cell biology to investigate what the proteins mutated in MKS do in cells. We have previously shown that cells where MKS proteins are mutated cannot make a cilium, but how this translates into the brain defects is unknown. We have hypothesised that this is because cells cannot migrate properly. During brain development, cells need to migrate to the correct position and if this goes wrong, there are many problems with the formation and organisation of the brain. We have shown that cells where MKS proteins are mutated cannot migrate well and the work that we propose here is aimed at understanding why this is and how MKS proteins function in cell migration. Understanding this is critical to understanding normal human development so that we can understand what goes wrong in disease, a pre-requisite for the design of treatments.

In recent years, many inherited diseases, of previously unknown aetiology, have been shown to be ciliopathies (diseases of cilium dysfunction) and there is considerable worldwide interest in elucidating the role of cilia in human development. Meckel-Gruber syndrome (MKS) is a lethal autosomal recessive disorder that represents the most common syndromic cause of neural tube defects in man; yet the reason for the neural tube defect is unknown. We and others have provided compelling evidence that MKS is a ciliopathy by showing that MKS proteins are required for centrosome polarisation during ciliogenesis, and this remains their only published role. However, MKS mutant cells also show aberrant actin polymerisation that is expected to have a profound effect on cell shape, polarity and migration. Neural tube defects can arise from defective cell migration or cell fate determination and our preliminary data demonstrate that there is a cell migration defect in MKS. The actin and microtubule cytoskeletons cooperate in polarised cell migration and understanding their mechanics in MKS is key to understanding the migration defect. How does cilium loss affect the organisation and dynamics of the actin cytoskeleton and cell adhesions? How do mutations in MKS proteins affect cell shape? How readily do MKS cells polarise towards a target? Why do MKS mutant cells migrate slower? Our overall aim is to use cell biology and genetics to give insight into how MKS proteins function in cell migration. We will image cell polarisation and formation and remodelling of actin networks and cell adhesions in MKS patient-derived immortalised fibroblast cell lines. We will use phase contrast and fluorescence microscopy of live and fixed cells in combination with assays designed to test the effect of mutations in MKS proteins on (a) initiation of polarity, (b) maintenance of polarity and directional cell migration, and (c) organelle polarisation and organisation of the actin cytoskeleton. Specifically, we aim to understand the following questions:

1. How do MKS fibroblasts behave during cell migration?

2. Are the dynamics and organisation of actin perturbed in MKS mutant cells?

These studies will increase our knowledge of the basic biology of ciliopathy, critical if these devastating disorders are ever to be treatable, and will facilitate further studies on the role of MKS proteins in migration in a developmental context.