Dissecting retrograde transport pathways in development and disease

© 2011 Dr. Pei Zhi Cheryl ChiaIntracellular trafficking pathways are required to transport proteins to different intracellular destinations. Some proteins undergo continuous shuttling between compartments and are known as recycling proteins. Two major sorting hubs are pivotal to the recycling of such cargoes: the early endosome and the trans-Golgi network (TGN). These two organelles mediate the major sorting decisions that determine the trafficking of different cargoes along distinct pathways. The aim of this thesis was to define the trafficking itineraries of a number of recycling proteins, which have previously been implicated in development and disease. These are 1) the ubiquitous endopeptidase furin, 2) the β-secretase BACE1 and 3) the multipass Wnt receptor, Wntless. Furin and the TGN-localized TGN38 are both membrane proteins that recycle between the TGN and plasma membrane. The Gleeson laboratory has previously shown that TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. In Chapter 3, the itinerary and transport requirements of furin have been identified. Using internalization assays, I show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were both required for efficient TGN-retrieval of furin. In contrast, TGN38 trafficking was independent of Rab9 and GCC185. This work highlights the existence of different retrograde pathways and the dependence on distinct subsets of trafficking machinery by different cargoes. Despite this, the determinants for the choice of a particular retrograde pathway remain poorly defined to date. In order to identify the sorting determinants for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated in Chapter 4. The diversion of furin from the Rab9-dependent late endosome-to-TGN pathway to the retromer-dependent early endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. Importantly, I present evidence to suggest that the length of the transmembrane domain could be a contributing factor in endosomal sorting. BACE1 cleavage of the amyloid precursor protein (APP) represents the initial step in the formation of the Alzheimer’s disease associated amyloidogenic Aβ peptide. Substantive evidence indicates that APP processing by BACE1 is dependent on intracellular sorting of this enzyme. Nonetheless, knowledge of the intracellular trafficking pathway of BACE1 remains fragmentary and previous studies have suggested that BACE1 recycles via the TGN. In Chapter 5, I show that cell surface BACE1 is rapidly internalized and transits the early endosomes en route to the Rab11-positive, juxtanuclear recycling endosome, with very little transported to the TGN as previously suggested. Moreover, BACE1 is predominantly localized to the recycling endosomal compartment in different cell types, including neurons. In contrast, very little APP trafficked to recycling endosomes. The influence of the trafficking itinerary of BACE1 on APP processing was also investigated. Using stable CHO cell lines, wild-type BACE1 was demonstrated to less efficient in Aβ production than a BACE1 chimera recycling via the TGN, highlighting the relevance of the itinerary of BACE1 on APP processing and the potential to manipulate the trafficking of BACE1 for therapeutic intervention. Based on these findings, I propose that the trafficking of BACE1 through the recycling endosomes transiently sequesters the enzyme from APP and contributes to the regulation of Aβ biogenesis in normal cells. The secretion of Wnt signalling proteins is dependent upon a transmembrane sorting receptor, Wntless (Wls), which recycles between the TGN and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. In Chapter 6, I demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, I have identified a conserved YXXɸ endocytosis motif at a novel position in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls-AEGL was rescued by the insertion of a classical YXXɸ motif in the cytoplasmic tail. Significantly, a Drosophila Wls-AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signalling. These findings demonstrate that YXXɸ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signalling proteins

Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

Recent improvements in the resolution of light microscopy, coupled with the development of a range of fluorescent-based probes, have provided new approaches to dissecting membrane domains and the regulation of membrane trafficking. Here, we review these advances, as well as highlight developments in quantitative image analysis and novel unbiased analytical approaches to quantitate protein localization. The application of these approaches to endosomal sorting and endosome-to-Golgi transport is discussed