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Paramecium tetraurelia basal body structure.

ABSTRACT: Paramecium is a free-living unicellular organism, easy to cultivate, featuring ca. 4000 motile cilia emanating from longitudinal rows of basal bodies anchored in the plasma membrane. The basal body circumferential polarity is marked by the asymmetrical organization of its associated appendages. The complex basal body plus its associated rootlets forms the kinetid. Kinetids are precisely oriented within a row in correlation with the cell polarity. Basal bodies also display a proximo-distal polarity with microtubule triplets at their proximal ends, surrounding a permanent cartwheel, and microtubule doublets at the transition zone located between the basal body and the cilium. Basal bodies remain anchored at the cell surface during the whole cell cycle. On the opposite to metazoan, there is no centriolar stage and new basal bodies develop anteriorly and at right angle from the base of the docked ones. Ciliogenesis follows a specific temporal pattern during the cell cycle and both unciliated and ciliated docked basal bodies can be observed in the same cell. The transition zone is particularly well organized with three distinct plates and a maturation of its structure is observed during the growth of the cilium. Transcriptomic and proteomic analyses have been performed in different organisms including Paramecium to understand the ciliogenesis process. The data have incremented a multi-organism database, dedicated to proteins involved in the biogenesis, composition and function of centrosomes, basal bodies or cilia. Thanks to its thousands of basal bodies and the well-known choreography of their duplication during the cell cycle, Paramecium has allowed pioneer studies focusing on the structural and functional processes underlying basal body duplication. Proteins involved in basal body anchoring are sequentially recruited to assemble the transition zone thus indicating that the anchoring process parallels the structural differentiation of the transition zone. This feature offers an opportunity to dissect spatio-temporally the mechanisms involved in the basal body anchoring process and transition zone formation.

SUBMITTER: Tassin AM

PROVIDER: S-EPMC4746876 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Project description:BackgroundThe development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane. In the multiciliated cell Paramecium, three conserved proteins, FOR20, Centrin 2, and Centrin 3 participate in this process, FOR20 and Centrin 2 being involved in the assembly of the transition zone. We investigated the function of two other evolutionary conserved proteins, OFD1 and VFL3, likely involved in this process.ResultsIn Paramecium tetraurelia, a single gene encodes OFD1, while four genes encode four isoforms of VFL3, grouped into two families, VFL3-A and VFL3-B. Depletion of OFD1 and the sole VFL3-A family impairs basal body docking. Loss of OFD1 yields a defective assembly of the basal body distal part. Like FOR20, OFD1 is recruited early during basal body assembly and localizes at the transition zone between axoneme and membrane at the level of the microtubule doublets. While the recruitment of OFD1 and Centrin 2 proceed independently, the localizations of OFD1 and FOR20 at the basal body are interdependent. In contrast, in VFL3-A depleted cells, the unanchored basal bodies harbor a fully organized distal part but display an abnormal distribution of their associated rootlets which mark their rotational asymmetry. VFL3-A, which is required for the recruitment of Centrin 3, is transiently present near the basal bodies at an early step of their duplication. VFL3-A localizes at the junction between the striated rootlet and the basal body.ConclusionOur results demonstrate the conserved role of OFD1 in the anchoring mechanisms of motile cilia and establish its relations with FOR20 and Centrin 2. They support the hypothesis of its association with microtubule doublets. They suggest that the primary defect of VFL3 depletion is a loss of the rotational asymmetry of the basal body which specifies the sites of assembly of the appendages which guide the movement of basal bodies toward the cell surface. The localization of VFL3 outside of the basal body suggests that extrinsic factors could control this asymmetry.

Project description:BackgroundA Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium.ResultsThe ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic tree, where P. tetraurelia sequences only partially cluster.ConclusionAnalysis of different features on nucleotide and amino acid level revealed striking differences in isoforms of actin and actin-related proteins in P. tetraurelia, both within the organism and in comparison to other organisms. This diversification suggests unprecedented specification in localization and function within a unicellular eukaryote.

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Paramecium tetraurelia basal body structure.

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