SummaryCentrioles are highly conserved structures that fulfil important cellular functions, such as nucleation of cilia and flagella (basal-body function) and organisation of pericentriolar material to form the centrosome. The evolution of these functions can be inferred from the distribution of the molecular components of extant centrioles and centrosomes. Here, we undertake an evolutionary analysis of 53 proteins known either for centriolar association or for involvement in cilia-associated pathologies. By linking protein distribution in 45 diverse eukaryotes with organism biology, we provide molecular evidence to show that basal-body function is ancestral, whereas the presence of the centrosome is specific to the Holozoa. We define an ancestral centriolar inventory of 14 core proteins, Polo-likekinase, and proteins associated with Bardet-Biedl syndrome (BBS) and Meckel-Gruber syndrome. We show that the BBSome is absent from organisms that produce cilia only for motility, predicting a dominant and ancient role for this complex in sensory function. We also show that the unusual centriole of Caenorhabditis elegans is highly divergent in both protein composition and sequence. Finally, we demonstrate a correlation between the presence of specific centriolar proteins and eye evolution. This correlation is used to predict proteins with functions in the development of ciliary, but not rhabdomeric, eyes.

SummaryTrypanosomes use a microtubule‐focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early‐branching eukaryote Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge‐like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α‐tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis‐positioning during cell division and cytokinesis defects.

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

X-linked severe combined immunodeficiency (SCID-X1), caused by a defect of the cytokine receptor common gamma chain (γc), has been successfully treated by gene therapy in the clinic. However, the occurrence of leukemia in several patients preceded by loss of oligoclonality revealed that treatment is associated with a risk inherent to the genetic modification of hematopoietic stem cells. In this study, we developed a safety approach that allows the specific elimination of gene-modified cells. For this, a small peptide sequence (myc-tag) was introduced into the murine γc protein. Cells expressing the modified chain can be detected with a myc-specific antibody by flow cytometry and are effectively depleted in vitro in the presence of complement factors. Further, thymic-derived T cells from mice reconstituted with myc-tagged γc-transduced bone marrow stem cells can be depleted by antibody administration in vivo. Similarly, specific complement-mediated lysis was observed for human T cells expressing the human myc-tagged γc. In a cell proliferation assay, the modified cytokine receptor chain showed no functional impairment compared to the wild-type chain. In sum, we show proof-of-principle of a safety mechanism for SCID-X1 gene therapy that would allow elimination of gene-corrected cells in a patient upon observation of monoclonal outgrowth.