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 centrioles surrounded by a matrix of pericentriolar material (PCM). The PCM is involved in nucleating microtubules and is composed of structural framework of 12-15 nm fibers termed the centromatrix. The centromatrix proper lacks microtubule-nucleating activity but binds g-tubulin ring complexes (g-TuRCs), lock washer-like structures that possess microtubule-nucleating and capping activities. 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 g-TuRC components 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. Previously we identified the protein ZYG-1 as a novel regulator of centrosome duplication in C. elegans. ZYG-1 is a kinase required for centriole assembly but the substrates of this kinase are currently unknown. To identify potential substrates and/or interacting factors we have carried out a genetic suppressor screen for mutations that restore viability to a zyg-1 mutant strain. To date, 41 independent suppressors have been identified and many of these mutations produce centrosome defects and embryonic lethality. We have mapped and tested almost all of these mutations by complemention assay. Our results indicate that a minimum of 15 new genes have been discoved. Currently we are working on several of the most interesting genes. We have cloned one of these genes and found that it encodes a protein involved in physically linking the centrosome and nucleus. Cytological analysis of embryos carrying the suppressor 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. My laboratory is also actively involved in studying the process of centrosome maturation and we have recently identified a key regulator named SPD-2. We find that SPD-2 is a coiled-coil protein that localizes to centrioles and PCM and is required for association of many factors with the centrosome. In the absence of SPD-2 the microtubule organizing capacity of the centrosome is greatly diminished and spindle assembly fails. We have also uncovered genetic interactions between SPD-2 and SPD-5 another coiled-coil protein required for maturation and between SPD-2 and the microtubule motor protein dynein. As SPD-5 is homologous to a vertebrate centromatrix component, we propose that SPD-2 and SPD-5 are components of the C. elegans centromatrix. Finally using a combination of genetics and cytology, we have uncovered a new role for ZYG-1 and SPD-5 in regulating anaphase spindle positioning.
| 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 |
