Rho GTPases Signalling and PAK Function During Zebrafish Development

Abstract

This thesis describes the use of zebrafish to study the role and mechanisms of Rho GTPase signaling. The primary focus has been the vertebrate- specific Cdc42-homology protein (Chp) and of the important downstream effector kinases PAKs. In epiboly, Chp is shown to be essential for the early cell surface localization of E-cadherin (E-cadh) and N2-catenin (N2-cat) at adherens junctions. Early development in zebrafish requires that embryonic tissues form by modulation of both cell adhesiveness and cell movement to orchestrate the complex processes of epiboly and gastrulation. Early on E-cadherin (E-cadh) mediated cell-cell adhesion functions in all cell layers. Regulatory mechanisms involving directed E-cadherin trafficking through the endosomal system have been invoked, but how this process is specifically modulated is not known. I have shown that loss of Chp, or expression of dominant inhibitory Chp, is accompanied by mis-localized E-cadh (with intact F- actin) prior to gastrulation. This signaling pathway involves activation of the ubiquitous effector kinase PAK, and involvement of the PAK-interacting exchange factor PIX. Loss of signaling by any of the three components results in the same underlying defects. These experiments provide the earliest known function for PAK1/2 in vertebrate development. Because the 'fragile-X syndrome" RNA binding proteins; FXR1 and FMR1 have been identified as direct binding partners for mammalian PAK1, I have also applied the zebrafish model to understand how this interaction applies to the role of FXR1 in muscle development. I have demonstrated that a loss-of-function FXR1 mutant (I304N) that fails to bind PAK1 is also unable to compensate for loss of Zf FXR1 in development suggesting the PAK1 kinase interaction is essential. The FXR1 KH(2) surface mutant Q348K/E352A is similarly defective. Binding of PAK1 to FXR1 (or FMR1) leads to phosphorylation of Ser-420 that is essential for FXR1 function. vi The functions of the Cdc42 target PAK4 are elusive due to its early embryonic lethal phenotype in mice. In a series of rescue experiments in zebrafish, I have shown that human PAK4 is able to compensate for loss of fish PAK4, and have been able to test what domains on PAK4 are essential for its early role in development. In these experiments it is clear that PAK4 needs to be cytoplasmic since a mutant nuclear localized PAK4 is non-functional. Detailed experiments reveal the importance of PAK4 in preventing cell apoptosis and for proper brain development in zebrafish embryos. I describe a series of experiments that address the importance of PAK4 binding sites for 14-3-3 and Cdc42. Overall these experiments demonstrate the utility of conducting loss-of-function experiments in a model vertebrate to understand the role of proteins that are either vertebrate-specific or are involved in signaling pathways that are not well conserved in lower organisms.