Golgi biogenesis and the Polo-like kinase in T. brucei

The Golgi apparatus lies at the crossroads of exocytic and endocytic membrane traffic in all eukaryotic cells. It receives the entire output of newly-synthesized proteins and lipids from the endoplasmic reticulum (ER), processes the covalently-bound oligosaccharides in the stack and then sorts them in the trans-Golgi network (TGN). From here they are secreted (either constitutively or in a regulated manner) via the plasma membrane or sent to the endosome/lysosome system. As with all other cellular organelles, the Golgi undergoes duplication and partitioning during the cell cycle, so as to ensure propagation through successive generations. Partitioning has been well-studied, mostly in mammalian cells, but studies on the duplication process have lagged behind, largely because of the complexity of this organelle in mammalian cells. Typically, these contain several hundred copies of the Golgi often subsumed within a ribbon-like structure, next to the nucleus and often the centrosomes. To study the duplication process we have turned to protozoan parasites, many of which typically have a single Golgi that duplicates during the cell cycle. In T. brucei, the causative agent of sleeping sickness in man and Nagana in cattle, we have shown that the old Golgi helps construct the new and that the new assembly site is dictated by a novel bilobe structure marked by TbCentrins2 and 4. Centrins are calmodulin-like, calcium binding proteins that are highly conserved and crucial for centrosome duplication and segregation. We can now add duplication of the Golgi (and associated ER exit sites) to this select list, and this proposal seeks to understand the mechanism by which this novel structure itself undergoes duplication. We have already made the pivotal discovery that the single polo-like kinase homolog in T. brucei (TbPLK) is essential for this process, and that it appears to operate in part through phosphorylation of TbCentrin2. We now seek to gain a deeper understanding of the precise mechanism. We will identify the TbPLK phosphorylation sites on TbCentrin2 and determine whether mutating them has an effect on bilobe and/or Golgi biogenesis. We will then identify the binding partners of TbPLK using the phosphopeptide binding module of the kinase, so as to understand how TbPLK is recruited to the bilobe and to find novel bilobe resident proteins. Finally, we will selectively inhibit TbPLK activity using a small molecule so as to permit studies of bilobe biogenesis in living cells.

This project clearly identified that the polo like kinase homolog in Trypanosoma brucei is an essential part of cell division is this important human pathogen. Using a variety of cell biological methods, we were able to show that the kinase was responsible for coordinating several vital steps in the assembly of the T. brucei cytoskeleton, which is tasked with giving the cell its shape and allowing it to swim inside its hosts. We made key contributions in explaining how and when the kinase migrates throughout the cell. We identified a key substrate protein that is phosphorylated by TbPLK, discovered when in the cell cycle this event occurred, and showed that blocking it had a direct impact on the ability of the cell to divide. Lastly, we devised a way to block the activity of TbPLK using a small molecule drug, which impeded cell division. This allowed us to show that drugs against TbPLK would be viable treatments for trypanosomiasis, which is a serious problem in sub-Saharan Africa and which few drugs are available.