Epithelial cells are polarized, meaning that the plasma membrane is divided into two separate surface areas, the apical and the basolateral domains. Other examples of polarized cell types are neurons (with axons and dendrites) and endothelial cells. In the epithelium, the basolateral domain faces the blood supply, where receptors bind and internalize nutrients and signaling molecules. The PGs at the cell surface are proteins with covalently attached long, linear and sulfated glycosaminoglycan (GAG; sugar) chains that support binding of proteins like growth factors to particular clusters of sulfate modification. Both the polymerization of GAGs and their sulfation take place in the lumen of the Golgi apparatus. The required substrates are UDP-sugars and nucleotide sulfate (PAPS) that are pumped from the cytoplasm and into the Golgi lumen through specific transporter proteins. We are particularly interested in the regulation of the availability of these substrates.
We have through our research suggested that the apical and basolateral secretory routes might segregate much earlier than stated in the textbooks (Prydz, Dick, Tveit 2008. How many ways through the Golgi maze? Traffic 9, 299-304). We have also recently demonstrated that reduced availability of PAPS in the Golgi lumen makes epithelial MDCK cells prioritize sulfation of a particular type of proteoglycans (PGs) called heparan sulfate (HS), allowing for more intense growth factor binding (Dick G et al. 2015). This occurs in a pathway not accessible to all PGs underway to the basolateral membrane. We wish to further study the underlying cellular mechanisms, because it is important to understand the contribution from proteoglycan co-receptors to growth stimulation by growth factors. We also wish to investigate the generality of this novel mechanism, also in relation to cancer cells and sulfate metabolism in general. The direction of the master work can be discussed.
We have both over-expressed and reduced the expression (siRNA) to manipulate the level of PAPS transporters in the Golgi membrane. Manipulation of a second PAPS transporter (PAPST2) by siRNA did not alter the PAPS uptake significantly. We are now in the process of applying the CRISPR/CAS technology to further study the role of these transporters.
Successful over-expression of the PAPS transporter-I seems to expand the Golgi apparatus in epithelial MDCK cells. Proteomic analysis is undertaken to address what proteins are co-induced in this situation. Along the same line, we wish to measure changes to the volume and membrane area of the Golgi apparatus upon changes in expression of PAPST-1. This work will be together with Frode Skjeldal.
We are also looking for an alternative substrate for PAPST2. In relation to this, we are also interested in methylation of proteins in the Golgi lumen, and uptake of the methyl donor SAM. This work is in collaboration with the Falnes group in the BMB section.
In collaboration with the group of Steven Wilson at Department of Chemistry, UiO we have established an analytical pipeline to quantify PAPS in biological samples by HPLC and mass spectrometry (MS), both in cell lysates and Golgi fractions, isolated by ultracentrifugation. This method is now used to address the mechanisms underlying PAPS synthesis in the cytoplasm and PG sulfation in the Golgi apparatus of epithelial cells.
Methods:
Methods mentioned above. In addition, general biochemical, molecular biology and cell biology methods. Take contact for further explanation.