The centrosome is the cell's primary microtubule-organizing center (MTOC). Through its ability to form a polarized array of microtubules, the centrosome participates in a number of critical cellular processes such as intracellular transport, the generation and maintenance of cellular polarity, cell motility, and cell division. To address questions concerning the assembly and function of the centrosome, we use the small genetically tractable nematode worm, Caenorhabditis elegans (C. elegans), as a model system. Because the mechanisms under study in C. elegans are likely to be highly conserved, our findings should prove valuable in understanding the analogous processes in humans and how defects in these processes lead to disease.
The centrosome participates in activities basic to cell function via its ability to nucleate the formation of microtubules, long linear polymers of tubulin that play a key role in the trafficking of materials within cells. The nucleation and anchoring of microtubules occurs within the outer structural component of the centrosome called the pericentriolar material (or PCM), a poorly defined meshwork of proteins. The PCM in turn is organized into a focus by the inner component, a pair of centrioles, which are barrel-shaped organelles with a conserved nine-fold symmetry. The amount of PCM determines the microtubule-nucleating capacity of the centrosome and the number of centriole pairs determines the number of centrosomes. Both of these parameters are controlled by the cell cycle. The goal of our research is to understand how cells control the number, assembly and function of centrosomes.
Centrosomes are major determinants of mitotic spindle bipolarity. A mitotic cell possesses two centrosomes, each of which establishes one of the spindle poles. During cytokinesis, the cleavage furrow bisects the spindle, ensuring that each daughter cell receives one centrosome. To assemble its own bipolar spindle, each daughter cell must duplicate its centrosome once—and only once. Defects in this process can lead to spindle abnormalities, aneuploidy, and ultimately genomic instability, a state associated with cells undergoing neoplastic progression.
We have identified three factors required for centrosome duplication. ZYG-1 is a protein kinase that localizes to centrioles. Mutations in the gene encoding ZYG-1 lead to embryonic lethality, marked by a failure to form bipolar spindles. The cause of this defect is a failure to assembly new (daughter) centrioles. Thus, ZYG-1 regulates the critical process of daughter centriole formation. To accomplish this task, ZYG-1 functions together with SPD-2, a coiled-coil protein. SPD-2 also localizes to the centrosome and functions to recruit ZYG-1 to sites of daughter centriole assembly. The third factor is protein phosphatase 2A, one of the major phosphatases in the cell. PP2A promotes proper levels of ZYG-1 and another centrosome duplication factor named SAS-5.
Utilizing genetic approaches, we have also identified factors that negatively regulate centrosome duplication. Among the factors that play inhibitory roles is SZY-20, a conserved centriole-associated RNA-binding protein that limits the amount of ZYG-1 at the centriole. Another layer of control is provided by the SCF E3 ubiquitin ligase complex and its substrate adaptor proteins LIN-23 and SEL-10. The SCF complex is required to maintain ZYG-1 protein at a low level and appears to function by promoting the degradation of ZYG-1. Most recently we have found that protein phosphatase 1-beta and its activators I-2 and SDS-22 also function to limit ZYG-1 protein levels. When PP1 activity is inhibited, ZYG-1 levels rise and supernumerary centrioles are produced. This leads to the formation of multipolar spindles and ultimately death. Further study of these factors will allow us to understand how the fidelity of centrosome duplication is achieved.
The centrosome is a dynamic organelle, and during the cell cycle, its composition changes dramatically. The change in composition is mirrored by changes in microtubule-nucleating capacity. Microtubule growth is initiated within the PCM. Early in the cell cycle, the centrosome contains only a small amount of PCM and organizes a relatively small number of microtubules. As cells progress toward mitosis, the amount of PCM and the microtubule-nucleating capacity increase, a process termed maturation. Maturation involves the recruitment of PCM components, such as gamma-tubulin from cytoplasmic stores, and has been intensively studied in embryonic systems, in which an initially inactive sperm-derived centriole pair must accrue PCM components from the oocyte cytoplasm to become a full-fledged MTOC.
Our lab has demonstrated that SPD-2 also functions in the process of maturation. In newly fertilized embryos, SPD-2 gradually accumulates at the centrosome concomitant with an increase in the number of microtubules organized. In embryos lacking SPD-2, the centrosomes accrue only small amounts of PCM, organize relatively few microtubules, and fail to assemble a mitotic spindle. SPD-2 is an unusual MTOC component in that it is required for both the assembly of centrioles and PCM. This suggests mechanistic similarities in the processes of duplication and maturation Further study of SPD-2 will provide important insight into the molecular mechanisms that underlie these essential processes.
Surprisingly, we have also found that ZYG-1 plays a role in regulating PCM assembly. In the absence of its negative regulator SZY-20, ZYG-1 levels at the centrioles increase two to three fold. Interestingly the levels of PCM around the centrioles also increase two to three fold. The increase in PCM levels is directly attributable to the elevated levels of ZYG-1 at the centrioles, as depletion of ZYG-1 can restore normal PCM levels in cells lacking SZY-20. Our results indicate that the processes of centriole assembly and PCM assembly share a common set of regulators. Further work will allow us to flesh out the molecular mechanisms underlying the complex regulatory mechanism that control the number and size of centrosomes and to understand how these processes go awry in disease.