Biochemistry and Pharmacology - Research Publications
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ItemBECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking(NATURE PORTFOLIO, 2024-02-20)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.
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ItemSpatial-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(WILEY, 2024-03)Alzheimer's disease is associated with increased levels of amyloid beta (Aβ) 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 β-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 Aβ secretion. A 20°C block in cargo exit from the Golgi confirmed β- and γ-secretase processing of APPswe in the Golgi. Inhibition of the β-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 Aβ production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aβ production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.
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ItemNo Preview AvailableThe endosomal system of primary human vascular endothelial cells and albumin-FcRn trafficking(The Company of Biologists, 2023-08-11)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.
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ItemNo Preview AvailableLong-term live cell imaging during differentiation of human iPSC-derived neurons(ELSEVIER, 2023-12-15)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.
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ItemNo Preview AvailableOrganelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures(CELL PRESS, 2023-07-25)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.
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ItemNo Preview AvailableArf5-mediated regulation of mTORC1 at the plasma membrane.(American Society for Cell Biology (ASCB), 2023-04-01)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.
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ItemNo Preview AvailableQuantification of Golgi Entry and Exit Kinetics of Protein Cargoes.(Springer US, 2023)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.
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ItemImmune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs(AMER SOC CLINICAL INVESTIGATION INC, 2022-11-15)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.
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ItemInteracting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down(WILEY, 2022-09)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.