Projects
Function of the Vertebrate Marginal Zone (VMZ)
We have identified and molecularly defined a hitherto uncharacterized membrane domain in epithelial cells, which we call the vertebrate marginal zone (VMZ) (find out more). The VMZ is composed of and generated by the Crumbs complex, one of the three major polarity complexes in epithelial cells. We have identified a number of proteins that localize to the VMZ and that directly interact with components of the Crumbs complex. Our goal is to elucidate the function and spatio-temporal control of this VMZ-associated protein network. In particular, we are interested in understanding how the VMZ controls HIPPO signaling and actin dynamics at the apical-lateral border, and how these processes are linked to epithelial cell polarization, morphogenesis, and apical membrane growth (find out more). To address this, we are applying a combination of loss- and gain-of-function approaches in 2D and 3D epithelial cultures, biochemical methods, proximity proteomics, and light and electron microscopy.
Dissecting epithelial polarity development in time and space
The establishment of epithelial apico-basal polarity critically depends upon the precise coordination of membrane trafficking, the control of the cell cytoskeleton, and the assembly of cell-cell junctions. Our goal is to elucidate the mechanisms that regulate the formation of a polarised epithelial phenotype in 2D and 3D epithelial cell culture models using quantitative and time-resolved proximity proteomics and correlative light and electron microscopy (CLEM). This will be achieved by tagging different polarity factors with the peroxidase APEX2, which offers a rapid labelling system to localise proteins by electron microscopy and to map their molecular environment by proximity biotinylation and quantitative mass spectrometry (find out more). This will produce important new insight into the spatio-temporal organisation and regulation of the epithelial polarity network.
Functional characterization of novel regulators of epithelial cell junctions
We have identified novel cell junction-associated proteins with potential roles in signaling, membrane trafficking, and the dynamic remodelling of the actin cytoskeleton. Our aim is to elucidate the functions and spatio-temporal control of such proteins in the context of epithelial polarity development and tight junction formation and plasticity. We are particularly interested in understanding how Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase activating proteins (GAPs) regulate the RhoGTPases cdc42, Rac1, and RhoA at epithelial cell junctions, using genetic approaches (CRISPR/Cas9 knockouts) and imaging (light and electron microscopy) in cultured cells and in model organisms (e.g. in Drosophila and zebrafish, in collaborations). In addition, we aim to produce mechanistic insight into protein function using biochemical and structural approaches (Xray and CryoEM).
Visualisation of epithelial cell architecture by correlative light and cryo electron tomography
Cell-cell junctions play fundamental roles in epithelial cells yet their native structure is unknown. We have established workflows for correlative light and cryo electron microscopy (cryoCLEM) to gain high-resolution structural information on epithelial cell architecture under close-to-native conditions. Epithelial cells grown on EM grids are vitrified, imaged by fluorescence light microscopy, and then “sliced” using cryo focused-ion-beam milling (cryo FIB-SEM) to produce thin (~200 nm) lamella suitable for transmission EM. Cellular structures exposed in such lamella are then imaged by cryo electron tomography and analysed. The EM facilities at NTU (SBS and AToM) are equipped with state-of-the-art instrumentation for cryoEM and CLEM, including a spinning disk microscope (Corrsight) dedicated for cryo fluorescence light microscopy, a cryo FIB-SEM (Aquilos), a 200 kV Arctica TEM, and a 300 kV Titan Krios TEM.
