Developmental Regulation of Centrosome Duplication

O'Connell, Kevin

Abstract

Centrosomes, which consist of a pair of centrioles wrapped within a cloud of pericentriolar material (PCM), are the primary microtubule-organizing centers (MTOCs) in most cells. In Caenorhabditis elegans, the kinase ZYG-1 is essential for the replication of centrosomes. Embryos lacking maternal ZYG-1 activity fail to replicate the paternally contributed centriole pair, and are thus unable to form bipolar spindles following first division. In contrast, loss of paternal ZYG-1 activity results in duplication failure during male meiosis, and the production of sperm with a single centriole. These sperm can still fertilize eggs but the resulting embryos assemble a monopolar rather than bipolar spindle at first division. Several recently published reports indicated that centrosome duplication might be regulated differently in different tissues. To investigate this in C. elegans, we have been analyzing the effect of zyg-1 mutations on centrosome duplication in the male and female germ lines. We have found that small truncations of the c-terminus of ZYG-1 cause a novel phenotype: the production of embryos with supernumerary replication-incompetent centrosomes. These embryos inherit up to eight centrioles that are unable to duplicate. We have found the extra centrioles arise solely from defects in the male germ line and that this phenotype behaves as a gain of function. In contrast, failure of mutant embryos to duplicate these centrosomes is due to a loss of function of maternal ZYG-1. Thus, a single mutation behaves as a loss-of-function allele in one developmental context and a gain-of-function allele in another, suggesting that ZYG-1 is subject to different forms of regulation in different tissues. Consistent with this, we find that these mutant forms of ZYG-1 localize normally to meiotic centrosomes but are almost absent from mitotic centrosomes. The low level of mutant ZYG-1 at mitotic centrosomes is not the result of an unstable protein, as the truncated forms of ZYG-1 are expressed at normal levels. To investigate the centrosome amplification phenotype further, we characterized centrosome behavior during meiosis in live and fixed spermatocytes. We found that centrosomes behave normally through the end of meiosis I but rapidly amplified as cells approached meiosis II. Amplification of centrosomes depends upon the centrosome duplication factor SAS-6 and additional genetic analysis indicates that most, if not all, of the extra centrosomes produced in the male germ line are structurally intact. We have confirmed this using transmission electron microscopy. In summary, our data indicate that centrosome duplication is regulated differently in mitosis and meiosis. To understand these differences at a molecular level, we are working to identify factors that provide mitotic or meiotic specific regulation of centrosome duplication. Animals just don't possess the ability to build a centrosome, they also possess the ability to destroy these organelles. In the female germline of many species, centrioles are eliminated during formation of the egg. In contrast, centrioles are maintained in the male germline so that a single centriole pair is packaged in the sperm. The zygote inherits the sperm centriole pair and thus begins life with the same centriole copy number as its parents. While the elimination program is essential for maintaining centriole number from one generation to the next, the molecular details of this process remain a mystery. We are currently testing our hypothesis that cellular pathways involved in the bulk degradation of cytoplasmic contents (autophagy and physiological apoptosis) play a role in centriole elimination. We have begun by characterizing the process of centriole elimination in wild-type germlines and find that it initiates during the late stages of oogenesis at a time when physiological apoptosis is known to be active. We are now working to develop additional markers that will be used to characterize the elimination program further. Once we complete our initial characterization, we will begin to examining the potential roles of autophagy and physiological apoptosis in centriole elimination. This will be accomplished by examining centriole behavior in germlines in which one or both degradation pathways are compromised by RNAi or existing mutants.