Adaptability of the Golgi-dependent secretory routes – GolgiPS

Human induced pluripotent stem cells (also known as hiPSCs) were chosen as the model for study. These cells can be differentiated into several cell types using protocols described in the literature or commercial kits. Thus, the cells before differentiation (pluripotent) and the differentiated cells have the same genetic background.

Using these models, the spatial organization of the Golgi apparatus was analyzed using fluorescent markers. The composition of proteins associated with the Golgi apparatus was analyzed quantitatively. Finally, real-time fluorescence microscopy was used to study the transport of reporter proteins.

As part of this project, the culture and manipulation of induced pluripotent stem cells were acquired, as well as their differentiation. Several hiPSC cell lines necessary for the experiments to be conducted were generated. Quality control of hiPSCs and validation of differentiated models were performed.

Analysis of the spatial organization of the Golgi apparatus in hiPSCs and in hiPSC-derived cardiomyocytes and chondrocytes showed differences in terms of distribution within the cell, reinforcing our hypothesis of an adaptation of the Golgi apparatus during cell differentiation.

The identification of proteins associated with the Golgi apparatus in hiPSCs and cardiomyocytes highlighted the specific presence or enrichment of certain proteins in a cell type.

The results obtained in the GolgiPS project confirm the hypothesis that the organization of the Golgi apparatus and its secretory functions adapt during cell differentiation.

Functional analyses will now need to be carried out to understand the precise role of the Golgi regulators identified in the homeostasis and functions of the Golgi apparatus, as well as their role in cell differentiation.

In the long term, the proteins identified in the GolgiPS project could represent molecular levers for the development of targeted therapies.

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