Biochemistry and Pharmacology - Research Publications
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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 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 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.