GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production.

The diversion of the membrane-bound β-site amyloid precursor protein-(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway.

Application of flow cytometry to analyze intracellular location and trafficking of cargo in cell populations.

Pulse shape analysis (PulSA) is a flow cytometry-based method that involves the measurement of the pulse width and height of a fluorescently labeled molecule simultaneously, enabling a multidimensional analysis of protein localization in a cell at high speed and throughput. We have used the method to detect morphological changes in organelles such as Golgi fragmentation, track protein trafficking from the cell surface, and also discriminate cells with different target protein localizations such as the Golgi, lyso-endosomal network, and the plasma membrane. Here, we describe the basic experimental setup and analytical methods for performing PulSA to examine membrane trafficking processes.

Membrane tethering.

Membrane trafficking depends on transport vesicles and carriers docking and fusing with the target organelle for the delivery of cargo. Membrane tethers and small guanosine triphosphatases (GTPases) mediate the docking of transport vesicles/carriers to enhance the efficiency of the subsequent SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion event with the target membrane bilayer. Different classes of membrane tethers and their specific intracellular location throughout the endomembrane system are now well defined.

TGFβ in T cell biology and tumor immunity: Angel or devil?

The evolutionally conserved transforming growth factor β (TGFβ) affects multiple cell types in the immune system by either stimulating or inhibiting their differentiation and function. Studies using transgenic mice with ablation of TGFβ or its receptor have revealed the biological significance of TGFβ signaling in the control of T cells. However, it is now clear that TGFβ is more than an immunosuppressive cytokine.

High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry.

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell.

Cargo trafficking between endosomes and the trans-Golgi network.

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.

Intracellular itinerary of internalised β-secretase, BACE1, and its potential impact on β-amyloid peptide biogenesis.

β-Secretase (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 internalised BACE1 remains in doubt.

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.

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling.

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network 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.

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein.

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention.

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin.

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin.

The regulation of endosome-to-Golgi retrograde transport by tethers and scaffolds.

Retrograde transport between endosomes and the trans-Golgi network (TGN) is essential for the recycling of membrane proteins which are involved in a range of biological processes. A variety of machinery components have been identified at the TGN which regulate endosome-to-TGN transport, including small G proteins, SNAREs, tethering factors and scaffold molecules. The challenge is to understand how these regulatory components orchestrate not only the specific docking and fusion of retrograde membrane carriers with the TGN, but also maintain the integrity of this highly dynamic compartment to ensure efficient delivery and export of cargo.