FcRn mediates fast recycling of endocytosed albumin and IgG from early macropinosomes in primary macrophages.

The neonatal Fc receptor (FcRn) rescues albumin and IgG from degradation following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here, we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin colocalises to SNX5-positive membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells.

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

The diversion of the membrane-bound ß-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 (Aß) 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. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aß production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aß production.

Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b- and AP4-dependent pathway.

The intracellular trafficking and proteolytic processing of the membrane-bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid-beta (Aß) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by ß-secretase (BACE1) and γ-secretase. Here, we show that the majority of newly synthesized APP is transported from the trans-Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post-Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aß secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aß.

Dysregulation of intracellular trafficking and endosomal sorting in Alzheimer's disease: controversies and unanswered questions.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid plaques in the brain consisting of an aggregated form of amyloid ß-peptide (Aß) derived from sequential amyloidogenic processing of the amyloid precursor protein (APP) by membrane-bound proteases ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase. The initial processing of APP by BACE1 is re-gulated by intracellular sorting events of the enzyme, which is a prime target for therapeutic intervention. GWAS (genome-wide sequencing studies) have identified several AD-susceptibility genes that are associated with the regulation of membrane trafficking, and substantial evidence now indicates that AD is likely to arise from defective membrane trafficking in either or both of the secretory and endocytic pathways. Considerable progress has been made in defining the intracellular trafficking pathways of BACE1 and APP and the sorting signals of these membrane proteins that define their itineraries. In this review we highlight recent advances in understanding the regulation of the intracellular sorting of BACE1 and APP, discuss how dysregulation of these trafficking events may lead to enhanced generation of the neurotoxic Aß products in AD and highlight the unresolved questions in the field.

Emerging Insights into the Roles of Membrane Tethers from Analysis of Whole Organisms: The Tip of an Iceberg?

Membrane tethers have been identified throughout different compartments of the endomembrane system. It is now well established that a number of membrane tethers mediate docking of membrane carriers in anterograde and retrograde transport and in regulating the organization of membrane compartments. Much of our information on membrane tethers have been obtained from the analysis of individual membrane tethers in cultured cells. In the future it will be important to better appreciate the network of interactions mediated by tethers and the potential co-ordination of their collective functions in vivo. There are now a number of studies which have analyzed membrane tethers in tissues and organisms which are providing new insights into the role of this class of membrane protein at the physiological level. Here we review recent advances in the understanding of the function of membrane tethers from knock outs (or knock downs) in whole organisms and from mutations in tethers associated with disease.

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. We illustrate in particular the application of PulSA for monitoring the trafficking of the membrane-bound enzyme furin and morphological changes to the Golgi caused by Brefeldin A.

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

ß-Secretase (BACE1) cleavage of the amyloid precursor protein (APP) represents the initial step in the formation of the Alzheimer's disease associated amyloidogenic Aß 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. Here we show that cell surface BACE1 is rapidly internalised by the AP2/clathrin dependent pathway in transfected cells and traffics to early endosomes and Rab11-positive, juxtanuclear recycling endosomes, with very little transported to the TGN as has been previously suggested. Moreover, BACE1 is predominantly localised to the early and recycling endosome compartments in different cell types, including neuronal cells. In contrast, the majority of internalised wild-type APP traffics to late endosomes/lysosomes. To explore the relevance of the itinerary of BACE1 on APP processing, we generated a BACE1 chimera containing the cytoplasmic tail of TGN38 (BACE/TGN38), which cycles between the cell surface and TGN in an AP2-dependent manner. Wild-type BACE1 is less efficient in Aß production than the BACE/TGN38 chimera, highlighting the relevance of the itinerary of BACE1 on APP processing. Overall the data suggests that internalised BACE1 and APP diverge at early endosomes and that Aß biogenesis is regulated in part by the recycling itinerary of BACE1.