Thompson Group - Oikopleura dioica Cell Cycle Variants
Results obtained from multiple knockouts of key mammalian cell cycle regulators combined with efforts at decomposing the complex cell cycle regulatory network into interlaced, yet somewhat independent oscillatory modules, are presenting new research opportunities in understanding evolution of the cell cycle itself and defining its links to developmental processes and differentiation. For example, experiments using mice and cell lines demonstrate cell division in the absence of S-phase CDK2, G1-phase CDK4/CDK6 and even upon simultaneous deletion of all interphase CDKs. Instead, CDK1 assumed these functions through association with interphase cyclins. Similar results have been obtained upon inactivation of cyclin E or deletion of all cyclin Ds, indicative of significant functional plasticity of cyclins and CDKs.
Functional plasticity in cell cycle regulation is evident in the numerous endoreduplicative cell cycle variants in a wide array of organisms from protists to humans, which may account for generating up to half of the world’s biomass. Despite this prevalence, endocycles remain poorly characterized at the molecular level. The transition to endocycles requires inhibition of CDK1 activity through premature activation of the CDK1-independent form of APC/C (Cdh1/Fzr), induction of CKIs and downregulation of mitotic cyclins. Progression through endocycles is then driven in part by cyclical alternate oscillations of APC/CCdh1, CKIs, CycE-CDK2, transcription factor E2F1, and the ubiquitin ligase CRL4. An unfortunate corollary of this evolutionary flexibility in cell cycle regulation is that polyploid nuclei can be genetically unstable and undergo error-prone mitoses. Untimely polyploidization is seen as a key early event in cancer ontogeny and in the generation of genetic diversity that render inoperable tumours more difficult to treat with successful outcomes.
O. dioica's cyclin and CDK complements reveal that the ´transcriptional´ regulators are similar to other complex invertebrates, whereas both the ´cell cycle ´ cyclin and CDK complements display amplifications centered on the cyclin D, cyclin B and CDK1 families. The cyclin D complement has quadrupled in size compared to most invertebrates and the variants display unique expression profiles that only partially overlap during somatic development. Cyclin Ds play central roles in relaying environmental stimuli to cell growth and cell cycle entry, mediated through inactivation of pRb. Extensive regulation of fundamental cyclin D domain features by alternative splicing taken together with the existence of multiple cyclin D variants with overlapping expression profiles is suggestive of an intricate rheostat relaying external cues to fine-tune growth, cell cycle entry and development. This is consistent with our observations that control of cell growth and ploidy during epithelial endocycling is primarily achieved through variation of G-phase length (Fig. 1).
Fig 1. Patterning of the oikoplastic epithelium of O. dioica through differential polyploidisation of the component cellular fields. Distinct fields grow through a cell type-specific number of endocycles, each characterised by progressive shortening of Gap phases (white arrows) separating successive S-phases (dark arrows). Final epithelial ploidy levels range from 34 C for the chain of pearls to over 1000 C for the giant cells of Fol and Eisen. Larger view
The most astonishing feature of the O. dioica CDK complement is the dramatic expansion of its CDK1 assemblage. Whereas not a single animal has been identified with more than one CDK1, O. dioica possesses five expressed paralogs. Despite high overall sequence identity between kinase domains of O. dioica CDK1 paralogs, proline substitutions are found in the activation T-loop and in the PSTAIRE helix, a critical cyclin interface. Amplification of CDK1 paralogs in O. dioica is unexpected, as suppression of CDK1 activity is central to endocycle transitions in Drosophila and mammals and OdCDK1 expression profiles do not match those of A- or B-type cyclins during somatic development. It is therefore conceivable that different odCDK1 paralogs preferentially interact with distinct cyclins, extending beyond the A- and B-type families. In such a scenario, the PSTAIRE and T-loop proline substitutions in odCDK1 paralogs could modulate the conventional inability of cyclin Ds to induce the active T-loop and PSTAIRE conformation upon binding to the cognate CDK. Additionally, the A4 to S4 substitution in the PSTA4IRE motif of all odCDK1 paralogs mimics the T4 residue in CDK4/6 homologs that directly interact with cyclin D. This alteration may enhance potential interaction of odCDK1s with D-type cyclins.
Somatic endocycles in O. dioica involve down regulation of cyclin B and A, with cyclin E levels retained, suggesting it may reprise roles of cyclin A in directing S-phase progression, as in both invertebrate, Drosophila, ovary and vertebrate, mammalian, TG endocycles. Interplay between cyclin D variants, involving sequential variant expression and cyclin D splicing, is likely to represent a catalyst of initiation, progression and termination of somatic endocycling, similar to cyclin D dynamics in mammalian TG and megakaryocyte endoreduplication. Amplification of the mitotic CDK1 family instead of the G1/S-phase regulating CDK2 or CDK6 families is surprising as O. dioica development is predominated by extensive endocycling and suppression of mitotic features. It will be intriguing to further evaluate responsiveness of individual odCDK1 paralogs to individual cyclin variants, CKIs, Cks1 and T-loop activation regulators during endocycling, mitosis and meiosis in light of their specific CDK1 sequence variations.