The Multiple Functions of EFHC1 in Tetrahymena and Xenopus

Ying Zhao, University of Colorado Boulder

Abstract

The basal body is the microtubule-organizing center that nucleates the axoneme of the cilium. Both basal bodies and cilia are complex intracellular organelles composed of hundreds of polypeptides found throughout the eukaryotes. Different types of cilia have various and often over-lapping functions, from driving fluid flow and cellular motility, to the establishment of distinct cellular domains and morphologies, to mediating and modulating intracellular signaling involved in tissue patterning. Dysfunction of basal body or ciliary components has been implicated in a variety of human diseases. EFHC1 is conserved from single cell alga to human beings. Members of this protein family contain three tandem DM10 domains and a putative EF-hand Ca2+ binding motif at the C terminal end. In some organisms, the EF-hand motif appears to be missing. Mutations of EFHC1 have been linked to juvenile myoclonic epilepsy (JME). The EFHC1 paralog, EFHC2, is also present in many species and mutant forms are reported to be pathological. Despite their wide spread conservation, the cellular functions of EFHC family proteins and the underlying mechanisms associated with the pathology of mutant forms remains obscure. In this study, both the ciliate Tetrahymena thermophila and the frog Xenopus laveis were used as distinct systems to investigate the functions of EFHC1. Here I have shown that there are two EFHC1-like genes in Tetrahymena. Both of them encode proteins that localize to the basal bodies and the cilia, and their localization is independent on each other. Cells lacking either individual gene do not exhibit obvious growth or basal body defects. However, both of them have structural defects, missing the MIP1a complex inside the axonemal doublet microtubules. Unlike Tetrahymena, only the EFHC1b in X.laveis was confirmed to be expressed. I have demonstrated XlEFHC1 was associated with both motile and immotile cilia. It is involved in ciliated cell formation and cilia genesis. Knockdown of XlEFHC1 by morpholino resulted in disrupted central nervous system patterning and deficient neural crest formation and migration. Reduction of XlEFHC1 leads to increased level of Wnt8a RNA, suggesting XlEFHC1 acts as an antagonist of canonical Wnt signaling. Together, this thesis work uncovered unexpected roles of EFHC1 in maintaining axoneme structure, in ciliogenosis and in regulating Wnt signaling pathway, which might well be relevant to the etiology of JME.