Philadelphia University + Thomas Jefferson University

Research Projects

Research Projects

Regulation of cell junction dynamics by membrane traffic

Regulation of cell junction dynamics by membrane traffic.

Figure 1: Scheme of Rab13-mediated trafficking of RhoA.

We found that the GTPase Rab13, which recycles tight junction proteins, facilitates the translocation of RhoA and its guanine exchange factor PLEKHG5/Syx from cell junctions to the cell leading edge (Wu et al., 2011; Figure 1). This implicates Rab13 in cell migration, a previously unknown function of this protein. We are investigating the in vivo function of Rab13 using a new mouse model harboring an EC-specific deletion of Rab13. We previously found that global deletion of Rab13 is embryonic lethal. Our recent data indicates that deletion of Rab13 upon vascular endothelial cadherin (Cdh5) expression in the mouse embryo is not embryonic lethal. We have recently started phenotyping the rab13fl/fl Cdh5 mouse.

High-throughput identification of genes involved in mediating the effects of junction disruptors and stabilizers on endothelial cell junctions.

Endothelial cell junctions

Figure 2: ECs grown on microcarrier beads and stained by a cytoplasmic (green) or nuclear (red) dyes.

We are leveraging CRISPR-dCas9 gRNA inhibitory and activating libraries in order to either silence or activate genes that code for proteins that are components of cell junction maintenance pathways. Our current focus is on thrombin, but we will pursue additional cell-junction modifying pathways, e.g. VEGF and angiopoietin. In order to generate sufficient coverage of the gRNA libraries, which code for all the annotated human genes, we miniaturized the permeability assay to 100-200 mm microcarrier beads. We use a fluorescently-conjugated probe that binds to the bead’s 1.    surface which is exposed when the thrombin causes disassembly of the junctions between ECs (Figure 2). The resulting fluorescence signal facilitates sorting the beads to separate those where the EC junction response to thrombin is inhibited. In collaboration with Drs. Leonard Edelstein from the Cardeza Center and Eric Londin from the Computational Medicine Center of Thomas Jefferson University, we will identify genes that are required for mediating the effect of thrombin on cell junctions.

The role of membrane trafficking in EC junction homeostasis.

project 3

Figure 3: Micro-computed tomography images of the arterial coronary systems in mice of the indicated genotypes.

Protein density in the cytoplasm next to EC junctions is one of the highest in the cell. These proteins, mostly organized in transient complexes, are constantly recycled in resting ECs, and translocate from and to the junctions when challenged by junction disruptors such as thrombin. Numerous junction transmembrane and cytoplasmic proteins are scaffolded by MPDZ, a large protein consisting of numerous protein-binding modules. We posit that MPDZ is the core of a large protein complex that changes its composition upon EC response to physiological cell disruptors. Our objective is to 


Figure 4: EC contours immunolabeled by antibody to vascular endothelial cadherin in retina vessels of mice of the indicated genotypes.

determine where this complex translocates to and how its composition changes during this process. We identified new binding partners of MPDZ that facilitate its membrane trafficking. In parallel, we are phenotyping a new Mpdz loss-of-function mouse model, to test the in vivo function of MPDZ. We found that the coronary (Fig. 3) and retina (Fig. 4) vessels are deformed in Mpdz-/- mice, and that EC junctions in the vessels of the retina are disorganized in comparison to wild type mice. Newborn Mpdz-/- mice on a C57BL/6J genetic background suffer from severe hydrocephalus and die prematurely. We are currently analyzing the mechanism responsible for this effect in collaboration with Dr. Richard Smeyne from Neuroscience. Brain tumors are often accompanied by intracranial edema, a condition that can be life threatening in itself. In collaboration with Dr. Craig Hooper from Cancer Biology, we are comparing the vasculature in brains and glioma tumors of wild type versus Mpdz-/-mice.