FcRn regulates antigen presentation in dendritic cells downstream of DEC205-targeted vaccines.

Dendritic cell (DC)-targeted vaccination is a new mode of antigen delivery that relies on the use of monoclonal antibodies (mAb) to target antigen to specific DC subsets. The neonatal Fc receptor (FcRn) is a non-classical Fc receptor that binds to immunoglobulin G (IgG) in acidified endosomes and controls its intracellular transport and recycling. FcRn is known to participate in the antigen presentation of immune complexes, however its contribution to DC-targeted vaccination has not previously been examined. Here we have investigated the role of FcRn in antigen presentation using antigen conjugated to IgG mAb which target specific DC receptors, including DEC205 and Clec9A expressed by the conventional DC 1 (cDC1) subset. We show that FcRn is expressed at high levels by cDC1, both at steady-state and following activation and plays a significant role in MHC I cross-presentation and MHC II presentation of antigens that are targeted to cDC1 via mAb specific for DEC205. This effect of FcRn is intrinsic to cDC1 and FcRn impacts the efficacy of anti-DEC205-mediated vaccination against B cell lymphoma. In contrast, FcRn does not impact presentation of antigens targeted to Clec9A and does not regulate presentation of cell-associated antigen. These data highlight a new and unique role of FcRn in controlling the immunogenicity of anti-DEC205-based vaccination, with consequences for exploiting this pathway to improve DC-targeted vaccine outcomes.

Spatial-Temporal Mapping Reveals the Golgi as the Major Processing Site for the Pathogenic Swedish APP Mutation: Familial APP Mutant Shifts the Major APP Processing Site.

Alzheimer's disease is associated with increased levels of amyloid beta (Aß) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the ß-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aß secretion. A 20°C block in cargo exit from the Golgi confirmed ß- and γ-secretase processing of APPswe in the Golgi. Inhibition of the ß-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aß production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aß production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

Long-term live cell imaging during differentiation of human iPSC-derived neurons.

Live-cell imaging is crucial to appreciate the dynamics and the complexity of cellular interaction processes. However, live-cell imaging of human neurons is challenging due to neuronal sensitivity. Here, we describe a long-term live-cell imaging protocol for neurons derived from human induced pluripotent stem cells. By using an IncuCyte live-cell imaging system, we have obtained information on neuronal dynamics during the different stages of neurogenesis. The protocol has also been developed to monitor the dynamics of the neuronal intracellular organelles. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

The endosomal system of primary human vascular endothelial cells and albumin-FcRn trafficking.

Human serum albumin (HSA) has a long circulatory half-life owing, in part, to interaction with the neonatal Fc receptor (FcRn or FCGRT) in acidic endosomes and recycling of internalised albumin. Vascular endothelial and innate immune cells are considered the most relevant cells for FcRn-mediated albumin homeostasis in vivo. However, little is known about endocytic trafficking of FcRn-albumin complexes in primary human endothelial cells. To investigate FcRn-albumin trafficking in physiologically relevant endothelial cells, we generated primary human vascular endothelial cell lines from blood endothelial precursors, known as blood outgrowth endothelial cells (BOECs). We mapped the endosomal system in BOECs and showed that BOECs efficiently internalise fluorescently labelled HSA predominantly by fluid-phase macropinocytosis. Pulse-chase studies revealed that intracellular HSA molecules co-localised with FcRn in acidic endosomal structures and that the wildtype HSA, but not the non-FcRn-binding HSAH464Q mutant, was excluded from late endosomes and/or lysosomes. Live imaging revealed that HSA is partitioned into FcRn-positive tubules derived from maturing macropinosomes, which are then transported towards the plasma membrane. These findings identify the FcRn-albumin trafficking pathway in primary vascular endothelial cells, relevant to albumin homeostasis.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures.

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

Arf5-mediated regulation of mTORC1 at the plasma membrane.

The mechanistic target of rapamycin (mTOR) kinase regulates a major signaling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localized at other locations. However, little is known about the recruitment and activation of mTORC1 at nonlysosomal sites. To identify regulators of mTORC1 recruitment to nonlysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signaling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2-enriched membrane ruffles together with the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor, and Arf5 was shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and refeeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

Quantification of Golgi Entry and Exit Kinetics of Protein Cargoes.

The Golgi apparatus is a pivotal secretory organelle in membrane trafficking, a hub responsible for posttranslational modifications, sorting, and trafficking of newly synthetized proteins received from the endoplasmic reticulum (ER). Different protein cargoes have been shown to travel through the Golgi stacks with different kinetics. Dysregulated transport and altered residency time of cargoes in the Golgi can impair their functionality. To study the anterograde trafficking of specific protein cargoes, innovative molecular methods have been developed to synchronize the traffic of selected cargoes from the ER in live cells. These methods of synchronization now provide the ability to quantify the Golgi entry and exit kinetics of defined cargo. In this chapter, we describe a quantitative, accurate, and semiautomated protocol to image and quantify the anterograde trafficking of individual cargo traversing the Golgi. This protocol, using free software, is compatible with different synchronization techniques, and can be used for a range of applications, such as comparing the Golgi kinetics of (1) different cargoes, (2) wild-type cargo vs mutated cargo, (3) the same cargo under different Golgi conditions, and (4) cargoes in drug screening platforms. The method can also be applied to study the localization and transit of a cargo through different organelles other than the Golgi apparatus.

Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs.

A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under nonhemophilic conditions was maintained by programmed death (PD) ligand 1-expressing (PD-L1-expressing) regulatory T cells (Tregs) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Tregs and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and reengaged removal of inhibitor-forming B cells. We also demonstrated the existence of FVIII-specific Tregs in humans and showed that such Tregs upregulated PD-L1 in patients with hemophilia after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1-mediated B cell tolerance against FVIII operated in healthy individuals and in patients with hemophilia A without inhibitors, and that ITI reengaged this mechanism. These findings may impact monitoring of ITI success and treatment of patients with hemophilia A.

Interacting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down.

The small G protein Arl5b is localised on the trans-Golgi network (TGN) and regulates endosomes-to-TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane-bound Arl5b(Q70L)-GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP-Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN-localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.

Segregation of the membrane cargoes, BACE1 and amyloid precursor protein (APP) throughout the Golgi apparatus.

The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques.

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG, and contributes to their long plasma half-life. We recently identified an FcRn-dependent recycling pathway from macropinosomes in macrophages; however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labeled human serum albumin (HSA-AF488) in BMDMs, and raster image correlation spectroscopy (RICS) analysis of single fluorescent-labeled albumin molecules to monitor the diffusion kinetics of internalized albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM-FRET following endocytosis, including detection of FRET in tubular-like structures. A nonbinding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.

Regulation of mTORC1 activity by the Golgi apparatus.

Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) is a major signalling kinase in cells that regulates proliferation and metabolism and is controlled by extrinsic and intrinsic signals. The lysosome has received considerable attention as a major hub of mTORC1 activation. However, mTOR has also been located to a variety of other intracellular sites, indicating the possibility of spatial regulation of mTORC1 signalling within cells. In particular, there have been numerous recent reports of mTORC1 activation associated with the Golgi apparatus. Here, we review the evidence for the regulation of mTORC1 signalling at the Golgi in mammalian cells. mTORC1 signalling is closely linked to the morphology of the Golgi architecture; a number of Golgi membrane tethers/scaffolds that influence Golgi architecture in mammalian cells that directly or indirectly regulate mTORC1 activation have been identified. Perturbation of the Golgi mTORC1 pathway arising from fragmentation of the Golgi has been shown to promote oncogenesis. Here, we highlight the potential mechanisms for the activation mTORC1 at the Golgi, which is emerging as a major site for mTORC1 signalling.

Amyloid ß production along the neuronal secretory pathway: Dangerous liaisons in the Golgi?

ß-amyloid peptides (Aß) are generated in intracellular compartments of neurons and secreted to form cytotoxic fibrils and plaques. Dysfunctional membrane trafficking contributes to aberrant Aß production and Alzheimer's disease. Endosomes represent one of the major sites for Aß production and recently the Golgi has re-emerged also as a major location for amyloid precursor protein (APP) processing and Aß production. Based on recent findings, here we propose that APP processing in the Golgi is finely tuned by segregating newly-synthesised APP and the ß-secretase BACE1 within the Golgi and into distinct trans-Golgi network transport pathways. We hypothesise that there are multiple mechanisms responsible for segregating APP and BACE1 during transit through the Golgi, and that perturbation in Golgi morphology associated with Alzheimer's disease, and or changes in cholesterol metabolism associated with Alzheimer's disease risk factors, may lead to a loss of partitioning and enhanced Aß production.

Sorting nexin 5 mediates virus-induced autophagy and immunity.

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.

A role for Rab30 in retrograde trafficking and maintenance of endosome-TGN organization.

Rab30 is a poorly characterized small GTPase. Here we show that Rab30 is localised primarily to the TGN and recycling endosomes in a range of cell types, including primary neurons; minor levels of Rab30 were also detected throughout the Golgi stack and early endosomes. Silencing of Rab30 resulted in the dispersal of both early and recycling endosomes and TGN compartments in HeLa cells. By analyzing cargo trafficking in Rab30-silenced and Rab30-overexpressing HeLa cells, we demonstrate that Rab30 plays a role in retrograde trafficking of TGN38 from endosomes to the Golgi, but has no apparent role in the endocytic recycling of the transferrin receptor to the plasma membrane. Five interactive partners with Rab30 were identified by pull-down and MS analysis using GFP-tagged Rab30 mutant, Rab30(Q68L). Two of the interactive partners identified were Arf1 and Arf4, known regulators of endosome to TGN retrograde transport. Knockdown of Arf1 and Arf4 results in GFP-Rab30 decorated tubules arising from the recycling endosomes, suggesting association of Rab30 with tubular carriers. Overall our data demonstrates a role for Rab30 in regulating retrograde transport to the TGN and maintenance of endosomal-TGN organization.

Local Secretory Trafficking Pathways in Neurons and the Role of Dendritic Golgi Outposts in Different Cell Models.

A fundamental characteristic of neurons is the relationship between the architecture of the polarized neuron and synaptic transmission between neurons. Intracellular membrane trafficking is paramount to establish and maintain neuronal structure; perturbation in trafficking results in defects in neurodevelopment and neurological disorders. Given the physical distance from the cell body to the distal sites of the axon and dendrites, transport of newly synthesized membrane proteins from the central cell body to their functional destination at remote, distal sites represents a conundrum. With the identification of secretory organelles in dendrites, including endoplasmic reticulum (ER) and Golgi outposts (GOs), recent studies have proposed local protein synthesis and trafficking distinct from the conventional anterograde transport pathways of the cell body. A variety of different model organisms, including Drosophila, zebrafish, and rodents, have been used to probe the organization and function of the local neuronal secretory network. Here, we review the evidence for local secretory trafficking pathways in dendrites in a variety of cell-based neuronal systems and discuss both the similarities and differences in the organization and role of the local secretory organelles, especially the GOs. In addition, we identify the gaps in the current knowledge and the potential advances using human induced pluripotent stem cells (iPSCs) in defining local membrane protein trafficking in human neurons and in understanding the molecular basis of neurological diseases.

Macropinocytosis in Different Cell Types: Similarities and Differences.

Macropinocytosis is a unique pathway of endocytosis characterised by the nonspecific internalisation of large amounts of extracellular fluid, solutes and membrane in large endocytic vesicles known as macropinosomes. Macropinocytosis is important in a range of physiological processes, including antigen presentation, nutrient sensing, recycling of plasma proteins, migration and signalling. It has become apparent in recent years from the study of specialised cells that there are multiple pathways of macropinocytosis utilised by different cell types, and some of these pathways are triggered by different stimuli. Understanding the physiological function of macropinocytosis requires knowledge of the regulation and fate of the macropinocytosis pathways in a range of cell types. Here, we compare the mechanisms of macropinocytosis in different primary and immortalised cells, identify the gaps in knowledge in the field and discuss the potential approaches to analyse the function of macropinocytosis in vivo.

The role of innate immune responses and neuroinflammation in amyloid accumulation and progression of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid beta (Aß) accumulation, tau pathology and neuroinflammation. Recently, there has been considerable interest in the role of neuroinflammation in directly contributing to the progression of AD. Studies in mice and humans have identified a role for microglial cells, the resident innate immune cells of the central nervous system, in AD. Activated microglia are a key hallmark of the disease and the secretion of proinflammatory cytokines by microglia may result in a positive feedback loop between neurons and microglia, resulting in ongoing low-grade inflammation. Traditionally, the pathways of Aß production and neuroinflammation have been considered independently; however, recent studies suggest that these processes may converge to promote the pathology associated with AD. Here we review the importance of inflammation and microglia in AD development and effects of inflammatory responses on cellular pathways of neurons, including Aß generation.