Cenexin1 and Odf2: splice variants with diverged cilium functions.

The primary cilium is an essential sensory “antenna” jutting from the cell surface of animal cells. Once thought to be a vestigial structure, the cilium has emerged in the last decade as a vital sensory organelle that impacts a broad spectrum of human diseases named ciliopathies.

The cilium contains a microtubule-based axoneme composed of nine doublet microtubules arranged in a radially symmetric pattern that grows from the distal end of the basal body. The basal body is a modified centriole, a structural component of the centrosome that contains a mother-daughter centriole pair. At ciliogenesis, the mother matures into a basal body for cilium assembly. The mother centriole is distinguished from the daughter by distal and subdistal appendages that are involved in basal body anchoring to the membrane prior to cilium assembly. Loss of Cenexin1, a component of the distal appendages, disrupts cilium assembly. Therefore, dissecting the components of basal bodies and determining how they work will unravel the molecular mechanisms of ciliogenesis, providing a deeper understanding of ciliopathies.

Outer dense fiber protein 2 (Odf2), discovered in the sperm tail cytoskeleton, is encoded by odf2, and is essential for several aspects of centrosome and cilium function. While odf2 knockout in mouse F9 cells did not display obvious mitotic spindle assembly or cell division defect, RNAi knockdown in HeLa cells disrupted mitotic spindle organization in a Polo-like kinase 1 (Plk1)-dependent manner. Moreover, knockout mice show a very early pre-implantation embryonic lethality. The odf2 gene encodes at least ten proteins by alternative splicing. Among these, at least two classes emerge: those that have a ~190 amino acid C-terminal extension and those that do not have this domain. Isoform 9, which has the C-terminal extension, is referred to as Cenexin1, whereas isoform 6, without the extension, is Odf2. This distinction is important, because the isoforms have different functions and patterns of subcellular localization. Odf2 is produced predominantly in the testis, where it localizes within flagella (cilia) at structures called outer dense fibers. Cenexin1, on the other hand, is a broadly expressed mother centriole-specific protein that localizes to the distal/subdistal appendages and is required for their assembly. These differences in localization suggest diverged functions for the splice variants at centrosomes and cilia.

In the March 15, 2013 issue of Cell Cycle, Kyung Lee’s lab used mouse F9 odf2 knockout cells to show that Cenexin1, but not its variant Odf2, is necessary and sufficient for primary cilium assembly. The odf2 cells, which express no endogenous Odf2 or Cenexin1, are deficient in assembly of primary cilia and lack distal/subdistal appendages. Chang and colleagues rescued cilium assembly in odf2 cells with Cenexin1 expression, but not with Odf2. While rescue of cilium assembly in odf2 cells was shown previously, the work from Lee’s lab shows that this function is specific to the Cenexin1 isoform. Regarding Odf2 function, work from another lab indicates that it is important for sperm morphogenesis, since about 50% of haploid sperm from odf2 heterozygous mutant mice have an overt “kinked” shape and disruption of the outer dense fibers.

Consistent with the role of Cenexin1 in ciliogenesis, Chang and colleagues show that it associates with Rab8a, a small G protein required for cilium assembly, in a GTP-dependent manner. This finding contrasts with a previous report that Odf2, rather than Cenexin1, bound to Rab8a. The basis of this contradiction is unresolved. In addition, the authors showed that centriolar recruitment of Chibby, a distal centriole protein required for ciliogenesis, requires Cenexin1 but not Odf2.

Chang and colleagues go on to show that even low expression of full-length Cenexin1, or a Cenexin1 mutant (S796A) defective in Plk1 binding could fully remedy ciliogenesis deficit in odf2 cells. However, neither Odf2 nor Cenexin1 deleted for its C-terminal extension rescued ciliogenesis. Consistent with these findings, immune electron microscopy revealed distal/subdistal appendage localization of Cenexin1 but not Odf2.

This study depicts a fascinating phenomenon, where, rather than having separate genes encode different proteins, splice variants from the same gene encode distinct functions and localization. In this case, Cenexin1 localizes to distal/subdistal appendages of basal bodies, whereas Odf2 goes to the axoneme. Future findings will do doubt further illuminate the disparate functions of the proteins encoded by odf2 in centrosome and cilium biology.