Septins From Protists to People

Septin GTPases form nonpolar heteropolymers that play important roles in cytokinesis and other cellular processes. The ability to form heteropolymers appears to be critical to many septin functions and to have been a major driver of the high conservation of many septin domains. Septins fall into five orthologous groups. Members of Groups 1–4 interact with each other to form heterooligomers and are known as the “core septins.” Representative core septins are present in all fungi and animals so far examined and show positional orthology with monomer location in the heteropolymer conserved within groups. In contrast, members of Group 5 are not part of canonical heteropolymers and appear to interact only transiently, if at all, with core septins. Group 5 septins have a spotty distribution, having been identified in specific fungi, ciliates, chlorophyte algae, and brown algae. In this review we compare the septins from nine well-studied model organisms that span the tree of life (Homo sapiens, Drosophila melanogaster, Schistosoma mansoni, Caenorhabditis elegans, Saccharomyces cerevisiae, Aspergillus nidulans, Magnaporthe oryzae, Tetrahymena thermophila, and Chlamydomonas reinhardtii). We focus on classification, evolutionary relationships, conserved motifs, interfaces between monomers, and positional orthology within heteropolymers. Understanding the relationships of septins across kingdoms can give new insight into their functions.

A split ribozyme that links detection of a native RNA to orthogonal protein outputs

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we used laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivered a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We resolved a thermodynamic model to guide RENDR design, showed how input signals can be transduced into diverse visual, chemical, and regulatory outputs, and used RENDR to detect an antibiotic resistance phenotype in bacteria. This work shows how transcriptional signals can be monitored in situ using RNA synthetic biology and converted into different types of biochemical information.

HAP2-Mediated Gamete Fusion: Lessons From the World of Unicellular Eukaryotes

Most, if not all the cellular requirements for fertilization and sexual reproduction arose early in evolution and are retained in extant lineages of single-celled organisms including a number of important model organism species. In recent years, work in two such species, the green alga, Chlamydomonas reinhardtii, and the free-living ciliate, Tetrahymena thermophila, have lent important new insights into the role of HAP2/GCS1 as a catalyst for gamete fusion in organisms ranging from protists to flowering plants and insects. Here we summarize the current state of knowledge around how mating types from these algal and ciliate systems recognize, adhere and fuse to one another, current gaps in our understanding of HAP2-mediated gamete fusion, and opportunities for applying what we know in practical terms, especially for the control of protozoan parasites.

Electron cryo-tomography structure of axonemal doublet microtubule from Tetrahymena thermophila

Doublet microtubules (DMTs) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMTs, collectively named the microtubule inner proteins (MIPs). We used cryoET to study axoneme DMT isolated from Tetrahymena. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP RIB72A/B binds to the luminal wall of DMT by multiple DM10 domains. We found FAP115, an MIP-containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of A-tubule. It contacts both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. We observed substantial structure heterogeneity in DMT in an FAP115 knockout strain, showing extensive structural defects beyond the FAP115-binding site. The defects propagate along the axoneme. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

Sex, amitosis, and evolvability in the ciliate Tetrahymena thermophila

Understanding the mechanisms that generate genetic variation, and thus contribute to the process of adaptation, is a major goal of evolutionary biology. Mutation and genetic exchange have been well studied as mechanisms to generate genetic variation. However, there are additional processes that may also generate substantial genetic variation in some populations and the extent to which these variation generating mechanisms are themselves shaped by natural selection is still an open question. Tetrahymena thermophila is a ciliate with an unusual mechanism of nuclear division, called amitosis, which can generate genetic variation among the asexual descendants of a newly produced sexual progeny. We hypothesize that amitosis thus increases the evolvability of newly produced sexual progeny relative to species that undergo mitosis. To test this hypothesis, we used experimental evolution and simulations to compare the rate of adaptation in T. thermophila populations founded by a single sexual progeny to parental populations that had not had sex in many generations. The populations founded by a sexual progeny adapted more quickly than parental populations in both laboratory populations and simulated populations. This suggests that the additional genetic variation generated by amitosis of a heterozygote can increase the rate of adaptation following sex and may help explain the evolutionary success of the unusual genetic architecture of Tetrahymena and ciliates more generally.

Atg5 Regulates Selective Autophagy of the Parental Macronucleus during Tetrahymena Sexual Reproduction

Nuclear autophagy is an important selective autophagy process. The selective autophagy of sexual development micronuclei (MICs) and the programmed nuclear degradation of parental macronucleus (paMAC) occur during sexual reproduction in Tetrahymena thermophila. The molecular regulatory mechanism of nuclear selective autophagy is unclear. In this study, the autophagy-related protein Atg5 was identified from T. thermophila. Atg5 was localized in the cytoplasm in the early sexual-development stage and was localized in the paMAC in the late sexual-development stage. During this stage, the degradation of meiotic products of MIC was delayed in atg5i mutants. Furthermore, paMAC was abnormally enlarged and delayed or failed to degrade. The expression level and lipidation of Atg8.2 significantly decreased in the mutants. All these results indicated that Atg5 was involved in the regulation of the selective autophagy of paMAC by regulating Atg8.2 in Tetrahymena.

Structures of outer-arm dynein array on microtubule doublet reveal a motor coordination mechanism

Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of Tetrahymena thermophila OAD arrays bound to MTDs in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide the MTDs. ATP hydrolysis in turn relaxes the TTH interfaces to effect free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.