Protein trafficking is the process through which proteins are transported to the cell surface, to an intracellular compartment or released in the extracellular medium. The route followed by these proteins within the cell is called the secretory pathway.
Newly synthesized cargo arrives from the endoplasmic reticulum (ER) into the Golgi, travels across the Golgi, and is sorted at the Trans Golgi Network (TGN) for transport to respective cellular compartments or for secretion by specific pleiomorphic transport carriers. Depending on the cell type, the newly synthesized transport carriers are targeted to apical and basolateral cell surface, early/sorting endosomes, late endosomes, recycling endosomes, secretory storage granules, preceding Golgi cisternae, or the ER (Figure on the right). Thus the selection and packaging of any given cargo at the TGN requires specific factors that will determine its final fate. This process of protein sorting at the TGN is therefore highly sophisticated and so far not very well understood.
The best characterized sorting event at the TGN thus far is that of lysosomal hydrolases. These proteins bind to the mannose-6-phosphate receptor (M6PR) and are transported to the lysosome by clathrin-coated vesicles. Additionally, integral membrane proteins destined to the cell surface are known to contain export signals in their cytoplasmic domains, but no general rule has emerged thus far for their export from the TGN.
1) How is secretory cargo sorted at the TGN?
Apart from the MP6R no cargo receptors have until now been identified for the sorting of soluble secretory cargo such as extracellular matrix proteins. How such molecules are sorted and packed into the budding transport carriers at the TGN remains unknown. However, recently we identified novel sorting components at the TGN that include actin, the actin severing proteins ADF/cofilin, the secretory pathway ATPase 1 (SPCA1) and Ca2+.
Our working hypothesis is that ADF/cofilin and actin concentrate SPCA1 molecules into a sorting domain, and this clustering is necessary for local accumulation of Ca2+ in the lumen of the TGN. Cargoes that have high affinity for Ca2+ are sequestered in this domain, which is subsequently separated by membrane fission to generate a cargo filled transport carrier for transport to the cell surface.
The major aim of the group is to understand the mechanism of how these components (actin, ADF/cofilin, SPCA1 and Ca2+) function in regulation of cargo sorting at the TGN.
2) How does impaired Ca2+ homeostasis in the TGN result in skin pathology?
Hailey-Hailey disease is an autosomal dominant skin disorder induced by the loss of one copy of the human ATP2C1 gene, encoding SPCA1. This disease is characterized by a disruption of cell-cell contacts (acantholysis) in the suprabasal layers of the skin. Desmosomal components such as desmocollins and desmogleins are synthesized normally but do not reach the cell surface. So far the mechanism of this disease remains elusive.
We aim to understand the sorting and trafficking of these components in the context of Hailey-Hailey disease.
To address these questions, we will integrate a wide range of techniques including cell and molecular biology, functional microscopy (FRET, FRAP, FLIM), in vitro reconstitution of cellular processes, biochemical analysis of protein sorting and protein-protein interactions. Furthermore we plan to develop a 3D cellular system to study polarized sorting using said techniques.