The S100 family of proteins are calcium-binding proteins expressed in a wide variety of tissues that participate in both intracellular and extracellular activities. One family member, S100P, has gained attention primarily in the context of promotion of carcinogenesis. Containing no enzymatic activity of its own, it interacts with multiple target proteins to regulate motility and invasion of cancer cells. Little is known about the role of S100P in trophoblast cells of the placenta, but our initial work has shown that it plays a crucial role in regulating both cellular motility and invasion. In this work, we sought to first establish the cellular localisation of S100P using different fractionation analysis. We first demonstrated the strictly cytoplasmic and membrane-associated localisation of S100P in different trophoblast cells including JEG-3, BeWo and HTR8, as well as cancer cells, regardless of the levels of S100P or any other changes such as excess calcium, addition of a non-ionic detergent, or the use of nuclear export inhibitors (Leptomycin B). Interestingly, tagging S100P with the fluorescent marker YFP led to a relocalisation of S100P from the cytoplasm/membrane fractions to nuclear fractions, suggesting the fusion protein had differential properties and cellular localisation. Further cellular compartmentalisation allowed us to determine that some of the S100P pools were also found in the plasma membrane fraction in both trophoblasts and in cancer cells. S100P was detectable at the extracellular surface using plasma membrane purification and biotinylation experiments. Analysis of the S100P structure suggests the presence of possible membrane-interacting residues. The C-terminal polybasic domain, in combination with potentially lipid-modified residues, may promote the association of S100P with negatively-charged membrane structures. Blockade of extracellular S100P through use of an S100P antibody or with cromolyn, a molecule shown to interact with helix 4 of S100P, results in decreases in both migration and invasion of choriocarcinoma cell line JEG-3, EVT-like HTR8/SVneo cells and EVT cells isolated from first trimester placenta, but did not lead to any changes in the formation of focal adhesions. This is in contrast to changes in total cellular levels of S100P in JEG-3 and HTR8 cells where a decrease in paxillin-containing focal adhesions can be seen. These studies therefore show that different pools of S100P, either intracellular or membrane-associated, promote trophoblast motility and invasion through two independent pathways. Further analysis in order to fully characterise the S100P interactome demonstrated the presence of S100P in high molecular weight complexes in trophoblast cells, and mass spectrometry analysis presented a number of proteins with increased abundance in S100P-expressing cells, although characterisation of specific S100P interactors remains to be fully explored.
|Date of Award||2020|
|Supervisor||Stephane Gross (Supervisor)|
- focal adhesions
- subcellular fractionation