Structural Characterization of ZYG-1 in Centriole Assembly

Centrioles are conserved microtubule-based structures present in all animal cells. They are essential for both centrosome formation and cilium biogenesis. While centrosomes are essential for cell division, cilia play important roles in signal transduction, cell development and homeostasis. Defective centrioles trigger apoptosis and cause a plethora of human diseases. Therefore, understanding the mechanisms governing centriole formation is of major importance to further functional characterizations of centrioles, centrosomes and cilia. During centriole duplication, daughter centrioles are formed from procentrioles. Five core centriolar proteins have been identified in nematodes. An assembly hierarchy of their incorporation into procentrioles has been established. One of the critical steps in centriole formation is the recruitment of the polo-like kinase ZYG-1/Plk4, the master regulator, by their centriolar receptors SPD-2/Asterless. Additionally, previous studies have shown that ZYG-1 is functionally auto-regulated in the cell. However, the molecular details of Plk4/ZYG-1 recruitment are not fully understood and the precise mechanism of the auto-regulation of ZYG-1 remains unknown. The aims of this project are, (1) to uncover whether the differential interactions of Plk4 with SPD-2 and Asterless in human are also conserved in other organisms; (2) to investigate the interplay between the different domains of ZYG-1. In practical terms, we will use combinatorial strategies that include the state-of-the-art techniques in structural studies, including X-ray crystallography, NMR, and electron microscopy, and functional assays in nematodes. The proposed research, once completed, will shed light on both the recruitment of the polo-like kinase Plk4/ZYG-1 during centriole assembly and the auto-regulation of ZYG-1 in the cell. Our structural studies are also expected to provide hints for therapeutic drug design targeting the critical kinase Plk4 in a long run.

Growth of all living lives is achieved by continuous division of their cells. One very important thing for such division to occur timely and faithfully is a tiny structure within the cell called the centrosome, which is only a thousandth of the width of a human hair. In the center of the centrosome are two perpendicularly arranged cylinder-like structures called centrioles. Despite the very small size of the centriole, it is actually the largest protein-based structure within the cell. Notably, generation of a new pair of centrioles has to be done before each cell division. To understand the complex structure of the centriole is like to solve a jigsaw puzzle but with more difficulties because of two reasons. First, as for now we still do not know exactly how many proteins are really in the centriole. Secondly, these "pieces" are so small that it is extremely difficult to "see" their structures. We aim to provide insights into these two aspects to understand the structure and function of centrioles. Through several years of extensive collaboration with the group of Dr. David Glover, a renowned geneticist at the University of Cambridge (moved to Caltech in 2019), we together revealed how a novel component of the centriole named Gorab works. Gorab was reported previously to be a resident protein at the Golgi, a critical membrane enclosed structure within all eukaryotic cells including humans. Our work demonstrates that Gorab actually has dual roles and shuttles between the Golgi and the centriole. In centrioles Gorab interacts with another centriolar protein Sas6 to stabilize the barrel-like structure, which is essential to ensure faithful cell division. Besides our published work on centrioles mentioned above, we have also characterized the structure of a protein named BILBO1, which is absolutely required for the human parasite Trypanosom brucei to survive. T. brucei is the causative agent of sleeping sickness that strikes millions of people in Africa. The atomic resolution crystal structure of a critical part of BILBO1 reveals an interesting horseshoe-like pocket that is the docking site of another parasitic protein. Our work sheds lights on a potential target for future therapeutic intervention to control sleeping sickness. Additionally, we have also identified and characterized a novel type of extended-synaptotagmins (E-Syts) in T. brucei. This protein serves to "stitch" the Golgi tightly to the membrane on the cell surface to allow active lipid shuttling between the two structures. Our work reals that the E-Syt protein of the parasite is the shortest known one of all known E-Syts, much shorter than all reported E-Syts from humans. We think that the unique feature of T. brucei E-Syt correlates with the early branching of the parasite during the evolutionary process of lives on earth.