SF-Assemblin genes in Paramecium: phylogeny and phenotypes of RNAi silencing on the ciliary-striated rootlets and surface organization

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

BackgroundMeckelin (MKS3), a conserved protein linked to Meckel Syndrome, assists in themigration of centrioles to the cell surface for ciliogenesis. We explored foradditional functions of MKS3p using RNA interference (RNAi) and expression of FLAGepitope tagged protein in the ciliated protozoan Paramecium tetraurelia.This cell has a highly organized cell surface with thousands of cilia and basalbodies that are grouped into one or two basal body units delineated by ridges. Thehighly systematized nature of the P. tetraurelia cell surface provides aresearch model of MKS and other ciliopathies where changes in ciliary structure,subcellular organization and overall arrangement of the cell surface can be easilyobserved. We used cells reduced in IFT88 for comparison, as theinvolvement of this gene’s product with cilia maintenance and growth is wellunderstood.ResultsFLAG-MKS3p was found above the plane of the distal basal body in the transitionzone. Approximately 95% of those basal bodies observed had staining for FLAG-MKS3.The RNAi phenotype for MKS3 depleted cells included global shortening andloss of cilia. Basal body structure appeared unaffected. On the dorsal surface,the basal bodies and their associated rootlets appeared rotated out of alignmentfrom the normal anterior-posterior rows. Likewise, cortical units were abnormal inshape and out of alignment from normal rows. A GST pull down using the MKS3coiled-coil domain suggests previously unidentified interacting partners.ConclusionsReduction of MKS3p shows that this protein affects development and maintenance ofcilia over the entire cell surface. Reduction of MKS3p is most visible on thedorsal surface. The anterior basal body is attached to and moves along thestriated rootlet of the posterior basal body in preparation for duplication. Wepropose that with reduced MKS3p, this attachment and guidance of the basal body islost. The basal body veers off course, causing basal body rows to be misalignedand units to be misshapen. Rootlets form normally on these misaligned basal bodiesbut are rotated out of their correct orientation. Our hypothesis is furthersupported by the identification of novel interacting partners of MKS3p including akinetodesmal fiber protein, KdB2.

Kinetoplast DNA (kDNA), the trypanosome mitochondrial genome, is a giant network containing several thousand interlocked DNA rings. Within the mitochondrion, kDNA is condensed into a disk-shaped structure positioned near the flagellar basal body. The disk is linked to the basal body by a remarkable transmembrane filament system named the tripartite attachment complex (TAC). Following kDNA replication, the TAC mediates network segregation, pulling the progeny networks into the daughter cells by their linkage to the basal bodies. So far TAC has been characterized only morphologically with no known protein components. By screening an RNAi library, we discovered p166, a protein localizing between the kDNA and basal body in intact cells and in isolated flagellum-kDNA complexes. RNAi of p166 has only small effects on kDNA replication, but it causes profound defects in network segregation. For example, kDNA replication without segregation causes the networks to grow to enormous size. Thus, p166 is the first reported molecular component of the TAC, and its discovery will facilitate study of kDNA segregation machinery at the molecular level.

Peter Satir (1936–2022), cell biology pioneer and mentor

Basal bodies nucleate, anchor, and organize cilia. As the anchor for motile cilia, basal bodies must be resistant to the forces directed toward the cell as a consequence of ciliary beating. The molecules and generalized mechanisms that contribute to the maintenance of basal bodies remain to be discovered. Bld10/Cep135 is a basal body outer cartwheel domain protein that has established roles in the assembly of nascent basal bodies. We find that Bld10 protein first incorporates stably at basal bodies early during new assembly. Bld10 protein continues to accumulate at basal bodies after assembly, and we hypothesize that the full complement of Bld10 is required to stabilize basal bodies. We identify a novel mechanism for Bld10/Cep135 in basal body maintenance so that basal bodies can withstand the forces produced by motile cilia. Bld10 stabilizes basal bodies by promoting the stability of the A- and C-tubules of the basal body triplet microtubules and by properly positioning the triplet microtubule blades. The forces generated by ciliary beating promote basal body disassembly in bld10Δ cells. Thus Bld10/Cep135 acts to maintain the structural integrity of basal bodies against the forces of ciliary beating in addition to its separable role in basal body assembly.

The surface pattern of Paramecium tetraurelia in interphase: an electron microscopic study of basal body variability, connections with associated ribbons and their epiplasmic environment

Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.

Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms.

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

Basal bodies are essential microtubule-based structures that template, anchor, and orient cilia at the cell surface. Cilia act primarily in the generation of directional fluid flow and sensory reception, both of which are utilized for a broad spectrum of cellular processes. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies of the ciliate Tetrahymena thermophila revealed important roles for two centrin family members in basal body assembly, separation of new basal bodies, and stability. Here, we characterize the basal body function of a centrin-binding protein, Sfr1, in Tetrahymena. Sfr1 is part of a large family of 13 proteins in Tetrahymena that contain Sfi1 repeats (SFRs), a motif originally identified in Saccharomyces cerevisiae Sfi1 that binds centrin. Sfr1 is the only SFR protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. In addition, Sfr1 resides predominantly at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Complete genomic knockout of SFR1 (sfr1Δ) causes a significant increase in both cortical row basal body density and the number of cortical rows, contributing to an overall overproduction of basal bodies. Reintroduction of Sfr1 into sfr1Δ mutant cells leads to a marked reduction of cortical row basal body density and the total number of cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study reveals an inhibitory role for Sfr1, and potentially centrins, in Tetrahymena basal body production. IMPORTANCE Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood. Despite the extensive study of centrins in Tetrahymena thermophila, little is known about how centrin-binding proteins contribute to centrin's roles in basal body assembly, stability, and orientation. The sole previous study of the large centrin-binding protein family in Tetrahymena revealed a role for Sfr13 in the stabilization and separation of basal bodies. In this study, we found that Sfr1 localizes to all Tetrahymena basal bodies and complete genetic deletion of SFR1 leads to overproduction of basal bodies. The uncovered inhibitory role of Sfr1 in basal body production suggests that centrin-binding proteins, as well as centrins, may influence basal body number both positively and negatively.

Basal bodies comprise nine symmetric triplet microtubules that anchor forces produced by the asymmetric beat pattern of motile cilia. The ciliopathy protein Poc1 stabilizes basal bodies through an unknown mechanism. In poc1∆ cells, electron tomography reveals subtle defects in the organization of intertriplet linkers (A-C linkers) that connect adjacent triplet microtubules. Complete triplet microtubules are lost preferentially near the posterior face of the basal body. Basal bodies that are missing triplets likely remain competent to assemble new basal bodies with nine triplet microtubules, suggesting that the mother basal body microtubule structure does not template the daughter. Our data indicate that Poc1 stabilizes basal body triplet microtubules through linkers between neighboring triplets. Without this stabilization, specific triplet microtubules within the basal body are more susceptible to loss, probably due to force distribution within the basal body during ciliary beating. This work provides insights into how the ciliopathy protein Poc1 maintains basal body integrity.

The freshwater planarian Schmidtea mediterranea has recently emerged as a valuable model system to study basal bodies (BBs) and cilia. Planarians are free-living flatworms that use cilia beating at the surface of their ventral epidermis for gliding along substrates. The ventral epidermis is composed of multiciliated cells (MCCs) that are similar to the MCCs in the respiratory airways, the brain ventricles, and the oviducts in vertebrates. In the planarian epidermis, each cell assembles approximately eighty cilia that beat in a coordinate fashion across the tissue. The BBs that nucleate these cilia all assemble de novo during terminal differentiation of MCCs. The genome of the planarian S. mediterranea has been sequenced and efficient methods for targeting gene expression by RNA interference are available. Defects induced by perturbing the expression of BB proteins can be detected simply by analyzing the locomotion of planarians. BBs are present in large numbers and in predictable orientation, which greatly facilitates analyses by immunofluorescence and electron microscopy. The great ease in targeting gene expression and analyzing associated defects allowed to identify a set of proteins required for BB assembly and function in planarian MCCs. Future technological developments, including methods for transgenic expression in planarians and in related species, will achieve turning free-living flatworms into powerful model systems to study MCCs and the associated human pathologies.

Basal body development and flagellar regression and growth in the unicellular green alga Chlamydomonas reinhardii were studied by light and electron microscopy during the vegetative cell cycle in synchronous cultures and during the sexual life cycle. Flagella regress by gradual shortening prior to vegetative cell division and also a few hours after cell fusion in the sexual cycle. In vegetative cells basal bodies remain attached to the plasma membrane by their transitional fibres and do not act as centrioles at the spindle poles during division. In zygotes the basal bodies and associated microtubular roots and cross-striated connexions all dissolve, and by 6·5 h after mating all traces of flagellar apparatus and associated structures have disappeared. They remain absent for 6 days throughout zygospore maturation and then are reassembled during zygospore germination, after meiosis has begun. Basal body assembly in developing zygospores occurs close to the plasma membrane (in the absence of pre-existing basal bodies) via an intermediate stage consisting of nine single A-tubules surrounding a central ‘cartwheel’. Assembly is similar in vegetative cells (and occurs prior to cell division), except that new basal bodies are physically attached to old ones by amorphous material. In vegetative cells, amorphous disks, which may possibly be still earlier stages in basal-body development occur in the same location as 9-singlet developing basal bodies. After the 9-singlet structure is formed, B and C fibres are added and the basal body elongates to its mature length. Microtubular roots, striated connexions and flagella are then assembled. Both flagellar regression and growth are gradual and sequential, the transitional region at the base of the flagellum being formed first and broken down last. The presence of amorphous material at the tip of the axoneme of growing and regressing flagella suggests that the axoneme grows or shortens by the sequential assembly or disassembly at its tip. In homogenized cells basal bodies remain firmly attached to each other by their striated connexions. The flagellar transitional region, and parts of the membrane and of the 4 micro tubular roots, also remain attached; so also do new developing basal bodies, if present. These structures are well preserved in homogenates and new fine-structural details can be seen. These results are discussed, and lend no support to the idea that basal bodies have genetic continuity. It is suggested that basal body development can be best understood if a distinction is made between the information needed to specify the structure of a basal body and that needed to specify its location and orientation.

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.

Immunofluorescence microscopy, conventional and high voltage transmission electron microscopy were used to describe changes in the flagellar apparatus during cell division in the motile, coccolithbearing cells ofPleurochrysis carterae (Braarud and Fagerlund) Christensen. New basal bodies appear alongside the parental basal bodies before mitosis and at prophase the large microtubular (crystalline) roots disassemble as their component microtubules migrate to the future spindle poles. By prometaphase the crystalline roots have disappeared; the flagellar axonemes shorten and the two pairs of basal bodies (each consisting of one parental and one daughter basal body) separate so that each pair is distal to a spindle pole. By late prometaphase the pairs of basal bodies bear diminutive flagellar roots for the future daughter cells. The long flagellum of each daughter cell is derived from the parental basal bodies; thus, the basal body that produces a short flagellum in the parent produces a long flagellum in the daughter cell. We conclude that each basal body in these cells is inherently identical but that a first generation basal body generates a short flagellum and in succeeding generations it produces a long flagellum. At metaphase a fibrous band connecting the basal bodies appears and the roots and basal bodies reorient to their interphase configuration. By telophase the crystalline roots have begun to reform and the rootlet microtubules have assumed their interphase appearance by early cytokinesis.