Lou Fourriere

Les microtubules (MTs) sont importants pour des processus cellulaires majeurs comme la polarisation, le trafic membranaire, la division cellulaire ainsi que pour l'architecture intracellulaire. En retour, les organites influencent l'organisation et la dynamique des MTs. Mon projet de thèse vise à élucider les interactions fonctionnelles existantes entre les MTs et la sécrétion en adoptant une approche quantitative et systématique. En synchronisant le trafic de différents cargos grâce au système RUSH (Retention Using Selective Hooks) et à une dépolymérisation complète des MTs, nous avons montré que les MTs ne sont pas strictement nécessaires à la sécrétion des cargos. D'une manière générale nous avons observé que le trafic intracellulaire est ralenti mais toujours possible en présence d'un appareil de Golgi dispersé. Nous avons caractérisé deux populations d'éléments golgiens. Elles sont présentes peu après la dépolymérisation des MTs et sont différentes en terme de composition et de capacité de sécrétion. Nos résultats montrent qu'une maturation fonctionnelle des éléments golgiens est nécessaire pour le trafic post-golgien, basée sur l'acquisition de certains facteurs golgiens non identifiés à ce jour. Dans une deuxième partie, nous nous sommes intéressés à l'exocytose au niveau de la membrane plasmique. Nous avons observé une sécrétion préférentielle des protéines à proximité des adhésions focales. Différentes techniques de biologie cellulaire nous ont permis de caractériser cet adressage préférentiel vers les sites d'adhésion de la cellule. Nous avons également corrélé les forces exercées par la cellule sur le substrat avec la direction du transport antérograde.

The production of neurotoxic amyloid-β peptides (Aβ) is central to the initiation and progression of Alzheimer's disease (AD) and involves sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are transmembrane proteins and their co-localisation in the same membrane-bound sub-compartment is necessary for APP cleavage. The intracellular trafficking of APP and the β-secretase, BACE1, is critical in regulating APP processing and Aβ production and has been studied in several cellular systems. Here, we summarise the intracellular distribution and transport of APP and its secretases, and the intracellular location for APP cleavage in non-polarised cells and neuronal models. In addition, we review recent advances on the potential impact of familial AD mutations on APP trafficking and processing. This is critical information in understanding the molecular mechanisms of AD progression and in supporting the development of novel strategies for clinical treatment.

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.

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. Key words Intracellular trafficking Golgi apparatus Quantification Synchronization RUSH

The intracellular trafficking of β‐site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid‐β 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 Aβ 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.

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, includingDrosophila, 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.

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.

Microtubules are part of the dynamic cytoskeleton network and composed of tubulin dimers. They are the main tracks used in cells to organize organelle positioning and trafficking of cargos. In this Review, we compile recent findings on the involvement of microtubules in anterograde protein transport. First, we highlight the importance of microtubules in organelle positioning. Second, we discuss the involvement of microtubules within different trafficking steps, in particular between the endoplasmic reticulum and the Golgi complex, traffic through the Golgi complex itself and in post-Golgi processes. A large number of studies have assessed the involvement of microtubules in transport of cargo from the Golgi complex to the cell surface. We focus here on the role of kinesin motor proteins and protein interactions in post-Golgi transport, as well as the impact of tubulin post-translational modifications. Last, in light of recent findings, we highlight the role microtubules have in exocytosis, the final step of secretory protein transport, occurring close to focal adhesions.

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.