Adaptability of the Golgi-dependent secretory routes – GolgiPS

Secretory protein transport is necessary to fulfil essential cellular functions. At the centre of the secretory pathway, the Golgi apparatus has to handle the diversity of the cargos to be transported. It is now clear that diversity in the Golgi-dependent secretory routes does exist and that multiple trafficking mechanisms are at work. However, if general regulators of secretory transport have been identified, specific regulators of the trafficking of cargos transported by a given specialized cell type are still unknown.
In the project GolgiPS, we will explore the adaptability of the secretory routes to fit to specific secretion needs. Cells from some organs will need to secrete rather small molecules while others need to secrete heavily glycosylated cargos. For instance, chondrocytes abundantly secrete components of the articular cartilage and thus have to sustain efficient transport of large proteins, which are the articular collagens. This means that more physiologically-relevant models have to be used compared to basic cellular models but keeping the feasibility of exploring intracellular compartments and employing genetic tools.
To study the adaptability of the Golgi-dependent transport routes, human induced pluripotent stem (iPS) cells will be differentiated into three cell types having distinct secretion needs, namely chondrocytes, cardiomyocytes and intestinal cells. Several features of Golgi-dependent processes will be analysed by comparison of the undifferentiated and differentiated iPS cells. The organization of the Golgi apparatus in terms of size, volume and dispersion will be assessed. The protein composition of the Golgi apparatus will be determined using proximity biotinylation coupled to quantitative mass spectrometry. This analysis will lead to the identification of key Golgi proteins regulated in the differentiated state. The level of these key Golgi proteins will be perturbed using genome editing and chemically-controlled protein degradation. Following depletion of the key proteins, functional analysis of the secretory capacities of the cells will be monitored. The transport of cargos will be quantitatively analysed using the RUSH (for Retention Using Selective Hooks) assay. We will measure the transport of a set of cargos of various size, topology or function. In addition, in a candidate approach, the transport of cargos specific to the cell type of interest will be quantitatively analysed.
This project will allow a better understanding of the adaptive capacity of the Golgi apparatus while specific secretion needs are required and will explore specialization of cellular functions upon cell differentiation