Search

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Ciliates, such as Tetrahymena thermophila, evolved complex mechanisms to determine both the location and dimensions of cortical organelles such as the oral apparatus (OA: involved in phagocytosis), cytoproct (Cyp: for eliminating wastes), and contractile vacuole pores (CVPs: involved in water expulsion). Mutations have been recovered in Tetrahymena that affect both the localization of such organelles along anterior-posterior and circumferential body axes and their dimensions. Here we describe BCD1 , a ciliate pattern gene that encodes a conserved Beige-BEACH domain-containing protein a with possible protein kinase A (PKA)-anchoring activity. Similar proteins have been implicated in endosome trafficking and are linked to human Chediak-Higashi syndrome and autism. Mutations in the BCD1 gene broaden cortical organelle domains as they assemble during predivision development. The Bcd1 protein localizes to membrane pockets at the base of every cilium that are active in endocytosis. PKA activity has been shown to promote endocytosis in other organisms, so we blocked clathrin-mediated endocytosis (using "dynasore") and inhibited PKA (using H89). In both cases, treatment produced partial phenocopies of the bcd1 pattern mutant. This study supports a model in which the dimensions of diverse cortical organelle assembly-platforms may be determined by regulated balance between constitutive exocytic delivery and PKA-regulated endocytic retrieval of organelle materials and determinants.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila., Competing Interests: The authors have declared that no competing interests exist.

Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases., (© 2018 Louka et al.)

The mechanisms that govern pattern formation within the cell are poorly understood. Ciliates carry on their surface an elaborate pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes by largely unknown mechanisms. Ciliates divide by tandem duplication: the cortex of the predivision cell is remodeled into two similarly sized and complete daughters. In the conditional cdaI-1 mutant of Tetrahymena thermophila , the division plane migrates from its initially correct equatorial position toward the cell's anterior, resulting in unequal cell division, and defects in nuclear divisions and cytokinesis. We used comparative whole genome sequencing to identify the cause of cdaI-1 as a mutation in a Hippo/Mst kinase. CdaI is a cortical protein with a cell cycle-dependent, highly polarized localization. Early in cell division, CdaI marks the anterior half of the cell, and later concentrates at the posterior end of the emerging anterior daughter. Despite the strong association of CdaI with the new posterior cell end, the cdaI-1 mutation does not affect the patterning of the new posterior cortical organelles. We conclude that, in Tetrahymena , the Hippo pathway maintains an equatorial position of the fission zone, and, by this activity, specifies the relative dimensions of the anterior and posterior daughter cell., (Copyright © 2017 by the Genetics Society of America.)

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Live imaging has become a powerful tool in studies of ciliary proteins. Tetrahymena thermophila is an established ciliated model with well-developed genetic and biochemical approaches, but its large size, complex shape, and the large number of short and overlapping cilia, have made live imaging of ciliary proteins challenging. Here we describe a method that combines paralysis of cilia by nickel ions and total internal reflection microscopy for live imaging of fluorescent proteins inside cilia of Tetrahymena. Using this method, we quantitatively documented the intraflagellar transport in Tetrahymena., (Copyright © 2015. Published by Elsevier Inc.)