Intracellular Transport: Engineering and Mechanisms (ITEM)

               We use the RUSH system which allows to synchronize cargo transport from the endoplasmic reticulum (ER) to the plasma membrane (Boncompain et al, 2012). We revealed that unexpectedly, microtubules are not needed for Golgi export but to enable fast functional maturation of the Golgi complex (Fourriere et al, 2020). We also showed that exocytosis occurs on restricted hotspots at the plasma membrane in a microtubule- and RAB6-dependent fashion (Fourriere, Kasri et al, 2019).

              We adapted the RUSH assay to High Content Screening to screen complex libraries of small molecules. We identified a small molecule inhibiting activation of ARF1 and ER export (Boncompain et al, 2019a). We also carried out differential screening and identified small molecules that reduce the transport of the co-receptor of HIV, the GPCR CCR5, to the cell surface. We further showed these molecules allow to reduce HIV infection of primary human macrophages (Boncompain et al, 2019b).

              We are currently investigating if there is a link between the topology of the ER and protein export. For this purpose, we are developing single-molecule tracking approaches linked to the RUSH assay. Because protein export from the ER is a very fast event, we are also implementing rapid ways to inactivate protein function in cells including auxin-based and other PROTAC-based systems. We are also analyzing how translocation into the ER influences further transport steps.

              How the Golgi apparatus organization and protein secretion undergo adaptation to fulfil specific secretion needs is still poorly understood. We are using human induced pluripotent stem (iPS) cells and induce their directed differentiation into three different cell types (chondrocytes, cardiomyocytes and hepatocytes), which display distinct secretion needs.

              The activity of the Golgi complex in transport processes can be modulated by applying internal and external forces, suggesting it behaves like a mechano-sensitive organelle (Guet et al, 2014; Romani et al, 2019; Matthieu Samuel PhD thesis, 2020). We are investigating underlying this mechano-sensitivity in 2D and 3D cultures. This work benefits from the development of new fluorescent probes (“Halo-Flippers”) to directly measure the tension of Golgi membranes.

              It is now well established that RAB6 regulates various transport pathways at the level of the Golgi complex (Goud et al, 2018). Most of studies on RAB6 function have been performed in vitro in cultured cells. To study its function in vivo, we have invested for several years in the generation of conditional RAB6 k/o mice (Cre-Lox system) (Bardin et al, Biol Cell 2015). Constitutive inactivation of the RAB6A gene (and thus depletion of the two ubiquitous isoforms RAB6A and RAB6A') is lethal at the embryonic stage (5.5 days post-coïtum). To achieve constitutive or inducible invalidation of RAB6A in specific cell lines and tissues, the "floxed" RAB6Astrain was crossed with mouse strains expressing the Cre recombinase gene under the control of several specific Cre promoters (Bardin and Goud, 2021).

              The depletion of RAB6A/A' has been investigated in the mammary gland luminal cells (Cayre et al, 2020), in CD4+ T cells (Carpier et al, 2018), and in the melanocyte lineage (Patwardhan et al, 2017). In neuronal cells, the depletion of RAB6A/A' does not result in a detectable phenotype but the double inactivation of RAB6A/A' and the neuronal isoform RAB6B results in pronounced microcephaly. This phenotype can be explained by the role of RAB6 in dynein-dependent transport of apical polarity proteins to the apical pole of neural stem cells (Brault et al, 2022). We are currently investigating the role of RAB6 in gut homeostasis by depleting the three isoforms in intestinal epithelial cells.

              The RAB6 KO mice are also used to investigate whether RAB6 GTPase could regulate a protumorigenic secretory pathway in breast cancer. For this project, the mice that we generated study the function of RAB6 in the mammary gland are crossed with a mouse model that develop within 5-6 months triple-negative breast tumors. We are currently monitoring whether RAB6 depletion delays or impairs tumor formation

        We kept on developing the recombinant antibody approach we initiated years ago. We notably created our own libraries based on proprietary nanobody scaffolds and used them both for cell biology studies and to pursue cancer-related objectives. We developed further our recombinant antibody approach and selected conformation-specific nanobodies to study dynamin and endocytosis (Galli et al, 2017). We also selected anti-DNAs that can be used to target tissues with DNA origami in vivo (Chakraborty et al, 2021). In parallel, we applied our approach to cancer-related questions and selected nanobodies against cell-surface antigens (Alijaj et al., 2020; Crepin et al, 2017a, 2017b; Moutel et al, 2020).

         We currently develop functional screening assays enabling the identification of nanobodies that can be used for specific cell targeting. We will use these approaches to identify nanobodies that can be used to arm CAR-T cells, to deliver toxic payloads to tumor cells or to label tumor cells in vivo This part of the project is under the supervision of Sandrine Moutel within the Therapeutic antibody facility linked to the team (https://curie.fr/plateforme/curiecoretech-anticorps-recombinants).

In addition to the use of nanobodies to develop novel diagnosis approaches and innovative treatments, we are also using our knowledge on trafficking control for translational applications. For example, he RUSH assay can be adapted to high content screening and we started to use it to develop translational projects related to CAR-T cells. We aim at identifying molecules able to block the transport of therapeutically relevant factors such as TNF, IL6, and PDL1 as we did before for CCR5 (Boncompain et al, 2019b). We also started to implement the use of the RUSH assay to control the release of functional -proteins in vivo in collaboration with G. Kroemer’s teams (Faculté des Cordeliers) (Liu et al, 2020). We also used it to control the transport and tune the activity of CAR proteins in T cells We will continue this development, improving control, exploiting stem cells and bringing the system to the animal. We will go beyond CAR-T and use the assay in vivo to control circuitry in the animal.