My laboratory is interested in how the microtubule cytoskeleton is organized to carry out its roles in a number of critical cellular processes such as the generation and maintenance of cell architecture and polarity, intracellular motility, and the assembly and positioning of the mitotic spindle. In many cell types, microtubules are organized by the centrosome, a specialized organelle composed of an orthogonal pair of centrioles surrounded by a matrix of pericentriolar material (PCM). During the cell cycle, the centrosome undergoes a number of structural transformations that are essential for its activity. Once per cell cycle, prior to mitosis, the centrosome duplicates. This event is of critical importance to mitotic spindle assembly as it ensures that two centrosomes are available to form the poles of the bipolar spindle. Duplication involves splitting of the existing centriole pair followed by the synthesis of a new centriole next to each old centriole. As the cell progresses toward mitosis, the microtubule nucleating capacity of the centrosome as well as the levels of centrosome-associated proteins increase, a process termed maturation. Despite the importance of centrosome duplication and maturation, little is known of how these processes are regulated at a molecular level. In my laboratory, we are using the small soil nematode Caenorhabditis elegans to study centrosome duplication and maturation. C. elegans has the advantages of strong genetic methodology and being amenable to cytological analysis. During the past year we have analyzed a newly identified set of genes that interact with zyg-1. ZYG-1 is an essential regulator of centrosome duplication, and in its absence, the embryonic cell divisions fail owing to the defect in bipolar spindle assembly. We have identified 39 independent suppressors that partially restore centrosome duplication and viability to animals carrying the zyg-1(it25) mutation. However, we find that none of these suppressors allow viability in the complete absence of zyg-1 suggesting that they work by one of two mechanisms. These mutations function by either increasing the activity of the mutant form of ZYG-1 or by reducing the cell's requirement for zyg-1. Using mapping, complementation testing, and genomic sequencing, we have determined that five of the mutations are intragenic (mutations in zyg-1) while the other 34 define 22 genes with putative roles in centrosome duplication. We have so far cloned three of these genes (two in the past year). The sun-1 gene was the first to be identified and encodes an inner nuclear envelope protein involved in anchoring the centrosome to the nucleus. Cytological analysis of embryos carrying the sun-1(bs12) mutation revealed that the normally close association of the nucleus and centrosome is often lost leading us to hypothesize that the centrosome-nucleus attachment complex negatively regulates centrosome duplication. We have now completed analysis of ZYG-12, another factor that participates in this anchoring mechanism and find that, unlike SUN-1, it does not appear to play a role in regulating duplication. Our results suggest the existence of a novel regulatory mechanism that involves the direct participation of sun-1. The two other suppressor genes identified appear to be involved in cell cycle control of centrosome duplication. One of these, szy-20, encodes a novel coiled-coil domain containing protein. We have preliminary results that indicate that SZY-20 is a centrosome component and that it regulates diverse aspects the cell division cycle. The other suppressor encodes an adapter for the anaphase-promoting complex (APC) which regulates progression through the cell cycle by targeting regulatory proteins for destruction. This result suggests a new role for the APC . We propose that the APC is involved in regulating centrosome duplication through regulated proteolysis. Finally, my laboratory is also actively involved in studying the process of centrosome maturation. Previously we uncovered a genetic interaction between zyg-1 and spd-5, a gene required for centrosome maturation. Unexpectedly, we find that these two genes appear to be involved in anaphase spindle positioning. We are currently using GFP tagging and live imaging to precisely define the nature of this role.
| Kemp, Catherine A; Song, Mi Hye; Addepalli, Murali Krishna et al. (2007) Suppressors of zyg-1 define regulators of centrosome duplication and nuclear association in Caenorhabditis elegans. Genetics 176:95-113 |
| Golden, Andy; O'Connell, Kevin F (2007) Silence is golden: combining RNAi and live cell imaging to study cell cycle regulatory genes during Caenorhabditis elegans development. Methods 41:190-7 |
| O'Connell, Kevin (2005) There's no place like WormBase: an indispensable resource for Caenorhabditis elegans researchers. Biol Cell 97:867-72 |
| Kemp, Catherine A; Kopish, Kevin R; Zipperlen, Peder et al. (2004) Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2. Dev Cell 6:511-23 |
