Figure 1. Electron micrograph of the Golgi apparatus with associated vesicles.
Secretory proteins, such as insulin, neurotransmitters, or extracellular matrix proteins, control the function of a multicellular organism. In humans, their mistargeting results in cancer, autoimmune disease, or type 2 diabetes (T2D). Secretory proteins are synthesized, glycosylated, folded, and quality checked in the endoplasmic reticulum (ER) before being loaded into carriers for transport to the Golgi apparatus. The molecules travel across the Golgi stacks and are sorted at the trans-Golgi Network (TGN). The TGN can be imagined as a post station where vesicular cargo carriers are addressed and sent to different destinations. Within cells, these molecules are either sorted toward intracellular compartments such as endosomes or secretory storage granules or to the cell surface for secretion (Pakdel and von Blume, 2018). Although the processes regulating the sorting of these proteins at the TGN have been the subject of intense research in the last three decades, the molecular mechanisms remain poorly understood (Blank and von Blume, 2017; Kienzle and von Blume, 2014; Ramazanov et al., 2021; von Blume and Hausser, 2019).
The major questions of our laboratory
How are secreted proteins sorted into a transport carrier?
The role of the mannose-6-phosphate cargo receptor in the delivery of soluble lysosomal hydrolases by clathrin-coated vesicles is well understood (Kornfeld and Mellman, 1989). However, we are only beginning to uncover the sorting mechanism of soluble secreted proteins such as extracellular matrix proteins.
This process requires the local influx of Ca2+ by the Ca2+ATPase SPCA1 localized in the TGN and is regulated by the actin cytoskeleton (von Blume et al., 2009, von Blume et al, 2011, Kienzle et al. 2014) (Kienzle et al., 2014; von Blume et al., 2011; von Blume et al., 2009). The local increase of Ca(2+) causes the Ca(2+)-binding protein Cab45 to cluster into oligomers which are fine-tuned by phosphorylation by Fam20C (Hecht et al., 2020) then, Cab45-cargo complexes are packaged into specific sphingomyelin (SM)-rich vesicles and transported to the plasma membrane for secretion (Crevenna et al., 2016; Deng et al., 2018).
Another Ca2+-binding protein called NUC-B1 recruits and sorts matrix metalloproteinases towards podosomes in the cis Golgi compartment. This process is essential to transport these matrix-degrading enzymes to podosomes to promote cell migration (Pacheco-Fernandez et al., 2020; Pacheco-Fernandez et al., 2021; Pakdel et al., 2021)
Figure 2. Schematic view of secretory cargo sorting at the trans-Golgi-Network (TGN). Schematic model depicts Cab45-dependent client sorting of secretory proteins (LyzC) into sphingomyelin-rich vesicles.
How is proinsulin sorted into secretory storage granules?
Despite insulin’s functional significance, our understanding of its sorting and packaging for secretion at the TGN remains poor. Proinsulin in -cells is synthesized in the ER and transported to the TGN to be sorted and packed into immature secretory storage granules (ISGs). In ISGs, proinsulin is proteolyzed to mature insulin by protein convertases PC1/3 and PC2. Mature insulin forms a hexameric crystal in the presence of Zn(2+). Responding to nutrient stimuli, mature SGs are mobilized to fuse with the plasma membrane and deliver insulin to the bloodstream. A significant challenge remains in understanding the nature of interactions between the drivers of sorting of insulin to SGs, largely due to the disconnect between the initial in vitro biochemical measurements and its relevance to the in vivo events. We decided to tackle this long-standing question in the field with a combination of biophysical and microscopic approaches.
Figure 3. Schematic view of secretory cargo sorting at the trans-Golgi-Network (TGN). Schematic representation of Ca(2+) and pH dependent 'sorting by aggregation' modell in insulin sorting cellls.
Methods used in the laboratory
We are applying classical cell biology methods using cultured cells. We also work with isolated pancreatic tissues, and patient samples derived, for instance, from T2D patients. In parallel, we intensively apply modern biochemistry approaches to reconstitute the protein complexes relevant for secretion. We use various fluorescence microscopy approaches such as laser scanning confocal microscopy, TIRF microscopy, and functional live-cell imaging approaches (FRAP, FRET, FLIM, RUSH) to visualize the processes. To investigate the ultrastructural contexts in cells, we use classical transmission- and immunoelectron microscopy and pan/expansion microscopy.
(Author names of lab members on papers and reviews in bold)
Blank, B., and J. von Blume. 2017. Cab45-Unraveling key features of a novel secretory cargo sorter at the trans-Golgi network. Eur J Cell Biol. 96:383-390.
Crevenna, A.H., B. Blank, A. Maiser, D. Emin, J. Prescher, G. Beck, C. Kienzle, K. Bartnik, B. Habermann, M. Pakdel, H. Leonhardt, D.C. Lamb, and J. von Blume. 2016. Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network. J Cell Biol. 213:305-314.
Deng, Y., M. Pakdel, B. Blank, E.L. Sundberg, C.G. Burd, and J. von Blume. 2018. Activity of the SPCA1 Calcium Pump Couples Sphingomyelin Synthesis to Sorting of Secretory Proteins in the Trans-Golgi Network. Dev Cell. 47:464-478 e468.
Hecht, T.K., B. Blank, M. Steger, V. Lopez, G. Beck, B. Ramazanov, M. Mann, V. Tagliabracci, and J. von Blume. 2020. Fam20C regulates protein secretion by Cab45 phosphorylation. J Cell Biol. 219.
Kienzle, C., N. Basnet, A.H. Crevenna, G. Beck, B. Habermann, N. Mizuno, and J. von Blume. 2014. Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting. J Cell Biol. 206:635-654.
Kienzle, C., and J. von Blume. 2014. Secretory cargo sorting at the trans-Golgi network. Trends Cell Biol. 24:584-593.
Pacheco-Fernandez, N., M. Pakdel, B. Blank, I. Sanchez-Gonzalez, K. Weber, M.L. Tran, T.K. Hecht, R. Gautsch, G. Beck, F. Perez, A. Hausser, S. Linder, and J. von Blume. 2020. Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs. J Cell Biol. 219.
Pacheco-Fernandez, N., M. Pakdel, and J. Von Blume. 2021. Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells. Bio Protoc. 11:e3936.
Pakdel, M., N. Pacheco-Fernandez, and J. von Blume. 2021. Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells. Bio Protoc. 11:e3958.
Pakdel, M., and J. von Blume. 2018. Exploring new routes for secretory protein export from the trans-Golgi network. Mol Biol Cell. 29:235-240.
Ramazanov, B.R., M.L. Tran, and J. von Blume. 2021. Sending out molecules from the TGN. Curr Opin Cell Biol. 71:55-62.
von Blume, J., A.M. Alleaume, G. Cantero-Recasens, A. Curwin, A. Carreras-Sureda, T. Zimmermann, J. van Galen, Y. Wakana, M.A. Valverde, and V. Malhotra. 2011. ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1. Dev Cell. 20:652-662.
von Blume, J., J.M. Duran, E. Forlanelli, A.M. Alleaume, M. Egorov, R. Polishchuk, H. Molina, and V. Malhotra. 2009. Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network. J Cell Biol. 187:1055-1069.
von Blume, J., and A. Hausser. 2019. Lipid-dependent coupling of secretory cargo sorting and trafficking at the trans-Golgi network. FEBS Lett. 593:2412-2427.
Figure 4. Liquid-liquid phase separation (LLPS) of a mutant of chromogranin B protein deficient of negatively charged amino acid stretches.