Julia von Blume, PhD

How are secretory proteins sorted and targeted to the cell surface?

Most secreted proteins contain a signal sequence that targets them to the endoplasmic reticulum (ER) during synthesis. There they are translocated into the ER lumen and once folded travel in coat complex II (COPII)-coated vesicles to the Golgi apparatus. After passage through the Golgi membranes, proteins are sorted at the TGN for transport to their final destination. Depending on the cell types these destinations include apical and basolateral cell surfaces, early/sorting endosomes, late endosomes, recycling endosomes, secretory storage granules and preceding Golgi compartments. Sorting of lysosomal hydrolases for delivery to pre-lysosomes is well established, the corresponding mechanism by which secreted proteins are sorted for transport to the plasma membrane is poorly understood.

• Ca2+ dependent sorting in the trans-Golgi Network
• The interconnection between Ca2+ dependent sorting and leukocyte trafficking
• Novel Ca²⁺ independent sorting pathways

In recent years we discovered a novel sorting mechanism of secretory proteins a process that had remained mysterious for decades. The major components regulating this process are F-actin, cofilin, the Golgi ATPase SPCA1, Ca2+, and Cab45. We reconstituted the binding of cofilin to SPCA1 that recruits F-actin to a specific domain to initiate a Ca²⁺ influx required for cargo sorting Next, we discovered how a Golgi resident protein Cab45 binds and sorts cargo molecules by selective Ca²⁺ dependent oligomerization. This discovery led to the critical concept of sorting of proteins independent of a classical sorting receptor. Our research focuses on the poorly understood sorting reactions in the trans-Golgi Network (TGN) with particular emphasis on the role of protein and lipid complexes that recognize and pack secreted proteins into specific transport carriers. This process is crucial to facilitate secretion and function of these proteins in different cell types and organisms. Moving forward, we plan to identify the molecular mechanisms facilitating Ca²⁺ dependent sorting applying a combination of cell-based experiments, high-end microscopy, and biochemical reconstitution assays. To further investigate the link between TGN dependent sorting and physiology such as diabetes, we will apply mouse models.

A second major research goal of the group is the investigation of the interconnection of Ca²⁺ dependent sorting and cell migration in leukocytes. Leukocyte trafficking positions immune cells in the body and therefore has a central role in the function of the immune system. The foundation of these studies is that Cab45 and SPCA1 are primary regulators of extravasation of leukocytes. Leukocyte extravasation requires the sorting and transport of a so far unknown specific set of soluble molecules in the TGN. We plan to identify and analyze the secretomes of primary leukocytes isolated from SPCA1 and Cab45 knock-out mice. Having identified these factors, we will reconstitute their sorting using biochemical assays to gain an insight into the molecular complex in the TGN that together with Cab45 steers this process. Moving forward, we will be able to monitor the significance of Cab45, SPCA1 and the new components for extravasation of leukocytes in living organisms using intravital microscopy. These experiments will give mechanistic insight into the importance of TGN sorting for macrophage extravasation and will thus explain the basic mechanism of innate immunity.

In a third approach, we investigate additional mechanisms of TGN based secretory cargo sorting. For instance, our unpublished work has shown that sorting of Matrix Metallo Proteinases (MMPs) to podosomes in macrophages occurs in independently of our Ca²⁺-dependent sorting. We have accomplished to identify novel sorting regulators required for the transport of MMP2 to podosomes. To monitor and investigate the role of a physiological system we generated estrogen-regulated Hoxb8 immortalized macrophages in which we use CRISPR/Cas9 deplete these components. This system will allow monitoring the trafficking of MMP2 in macrophages and will also enable to track their movement in living organisms. These data will give an insight into the relevance of sorting of proteins inside macrophages and will allow testing their role in matrix invasion in normal and cancer tissues.