Abstract
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.
Highlights
The development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane
Identification of the variable flagellar number mutant 3 (VFL3) and oro-facial-digital syndrome 1 protein (OFD1) genes in Paramecium tetraurelia While a single VFL3 gene is present in Chlamydomonas, four VFL3 genes (VFL3-1, VFL3-2 VFL3-3, and VFL34) are encoded in the P. tetraurelia genome which has undergone at least three successive whole-genome duplications (WGD) during the evolution [27]
Phylogenetic analysis indicates that the four P. tetraurelia genes can be grouped into two families (VFL3-A and VFL3-B), the VFL3-A proteins being more closely related to the Chlamydomonas one (Additional file 2: Figure S2)
Summary
The development of a ciliary axoneme requires the correct docking of the basal body at cytoplasmic vesicles or plasma membrane. Absent in land plants or higher fungi, one or several cilia involved in motility, feeding, sensation, and sexual process, localize at the cell surface of numerous protists This diversity is observed in metazoa in which the number of cilia protruding from the surface depends on the cellular type: either a solitary cilium, called primary cilium or several, often hundred cilia, can Depending on the cellular type, the development of the primary cilium is initiated in the cytoplasm after docking of the mother centriole, via its distal appendages to Golgi derived vesicles or, directly in the external cellular space, after docking at the plasma membrane [1, 2]. These structures cap the tip of the basal body before its docking at the cell surface [7]
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