Enrique Rodriguez-Boulan Laboratory

Research Interests:

Epithelial Cell Biology, Retinal Cell Biology, Age Related Macular Degeneration

The organization of polarized epithelia depends on an Epithelial Polarity Program (EPP) that utilizes three machineries, dynamically interplaying with each other, to build an epithelial cell (Figure 1). The Polarized Trafficking Machinery is an adaptation of the secretory and endocytic systems that are found in every cell to the task of sorting and delivering PM proteins and lipids to Apical and Basolateral PM domains. The Domain-Identity Machinery adapts a highly conserved set of polarity proteins and lipids that perform a variety of polarity functions across tissues and species, to the task of generating and maintaining the "identity" of the apical and basolateral domains. This "identity" task involves two major subtasks. The first one is to control the assembly of a circumferential belt at the top of the cell, Tight Junctions (TJ) (Septate Junctions in Drosophila), that provides a fence between the apical and basolateral domains and a selective para-cellular gate between the luminal compartment and the internal medium. The second one is to organize the secretory and endocytic systems and the cytoskeleton into a Polarized Trafficking Machinery that sorts and delivers proteins to apical and basolateral domains. This involves the adaptation of ancestral endocytic signals and decoding mechanisms to perform apical-basolateral sorting in the biosynthetic and recycling pathways. The 3D-Organization Machinery adapts a system of small GTPases that controls cell growth and cytoskeleton organization in every cell to the task of coordinating extracellular cues sensed by cell-cell and cell-substrate adhesion molecules with the Polarized Trafficking and Domain-Identity machineries to produce a polarized epithelial cell.

The introduction of the MDCK model 3 decades ago (Cereijido et al., 1978; Rodriguez-Boulan & Sabatini, 1978) allowed the mapping of the biosynthetic and recycling routes of apical and basolateral plasma membrane (PM) proteins (Figure 2) (reviewed in Rodriguez-Boulan et al., 2005). Apical and basolateral PM proteins are synthesized at the Endoplasmic Reticulum, transported to the Golgi complex and sorted at the Trans Golgi Network (TGN) into distinct apical and basolateral vesicular routes. After arrival at the cell surface, apical and basolateral membrane proteins are internalized into separate apical sorting endosomes (ASE) and basal sorting endosomes (BSE) (Figure 2) (Rodriguez-Boulan et al., 2005a), mixed in common recycling endosomes (CRE) and sorted into distinct recycling routes back to their original PM domain. The apical recycling route includes the Apical Recycling Endosome (ARE). More recent work has shown that some newly synthesized PM proteins move from TGN to CRE and are sorted there in not fully polarized MDCK cells.

To study the sorting and intracellular routes of PM proteins, we use a combination of molecular, cell biological and physiological techniques. Recently, we have been using quantitative microscopy and live imaging techniques for this purpose. For example, we microinject cDNAs encoding apical or basolateral plasma membrane (PM) and study how they are sorted at the Golgi complex and transported to the cell surface. Movie 1, from Deborde et al. 2008), shows how Neural Cell Adhesion Molecule (NCAM) coupled to GFP leaves the Golgi complex (marked with sialyl transferase-RFP, a marker for the trans Golgi network or TGN)

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The movie also shows how knock-down of Clathrin delays significantly the exit of NCAM-GFP from the TGN. Our laboratory is now continuing to characterize the involvement of clathrin and different families of clathrin adaptors in the sorting process.