Epsilon-tubulin is an essential component of the centriole

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains epsilon-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to epsilon-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, epsilon-tubulin is encoded by the BLD2 allele and epsilon-tubulin plays a role in basal body/centriole morphogenesis.     

Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

Basal bodies and centrioles play central roles in microtubule (MT)‐organizing centres within many eukaryotes. They share a barrel‐shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo‐tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo‐atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non‐tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ‐tubulin and other components participate in the assembly of the basal body.     

Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole

Centrioles/basal bodies have a characteristic cylindrical structure consisting of nine triplet microtubules arranged in a rotational symmetry. How this elaborate structure is formed is a major unanswered question in cell biology [1, 2]. We previously identified a 170 kDa coiled-coil protein essential for the centriole formation in Chlamydomonas. This protein, Bld10p, is the first protein shown to localize to the cartwheel, a 9-fold symmetrical structure possibly functioning as the scaffold for the centriole-microtubule assembly [3]. Here, we report results by using a series of truncated Bld10p constructs introduced into a bld10 null mutant. Remarkably, a transformant (DeltaC2) in which 35% of Bld10p at the C terminus was deleted assembled centrioles with eight symmetrically arranged triplets, in addition to others with the normal nine triplets. The cartwheels in these eight-membered centrioles had spokes approximately 24% shorter than those in the wild-type, suggesting that the eight-triplet centrioles were formed because the cartwheel's smaller diameter. From the morphology of the cartwheel spoke in the DeltaC2 centriole and immunoelectron-microscope localization, we conclude that Bld10p is a major spoke-tip component that extends the cartwheel diameter and attaches triplet microtubules. These results provide the first experimental evidence for the crucial function of the cartwheel in centriolar assembly.     

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission

Abstract One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine‐fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine‐fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro‐basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.     

OBSERVATIONS ON THE CENTRIOLES OF THE SEA URCHIN SPERMATOZOON

The spermatozoon of Lytechinus variegatus has two parallel centrioles. The basal body of the flagellum consists of the proximal centriole (a short cylinder of nine tubule-triplets) and its distal extension of nine tubule-doublets. The distal centriole lies near the distal end of the basal body, between the nucleus and the mitochondrion. The observations suggest that both the proximal and the distal centrioles are polarized structures, their tubule-triplets pitched in the same direction and their distal ends associated with the flagellar axoneme and with the mitochondrion, respectively. The distal centriole in different spermatozoa occupies different positions around the basal body-flagellum complex.     

Long-lost relatives reappear: identification of new members of the tubulin superfamily

The identification and analysis of new members of the tubulin superfamily has advanced the belief that these tubulins play important roles in the duplication and assembly of centrioles and basal bodies. This idea is supported by their distribution in organisms with centrioles containing triplet microtubules and by recent functional analysis of the new tubulins. delta- and epsilon-tubulin are found in most organisms that assemble triplet microtubules. delta-tubulin is needed for maintaining triplet microtubules in Chlamydomonas and Paramecium. epsilon-tubulin is needed for centriole and basal body duplication and is an essential gene in Chlamydomonas. The distribution of eta-tubulin is more limited and has been found in only four organisms to date. Phylogenetic analysis suggests that it is most closely related to delta-tubulin, which suggests that delta- and eta-tubulin could have overlapping functions.     

Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii

In eukaryotic cells, basal bodies and their structural equivalents, centrioles, play essential roles. They are needed for the assembly of flagella or cilia as well as for cell division. Chlamydomonas reinhardtii provides an excellent model organism for the study of the basal body and centrioles. Genes for two new members of the tubulin superfamily are needed for basal body/centriole duplication. In addition, other genes that play roles in the duplication and segregation of basal bodies are discussed.     

Basal Body Assembly in Ciliates: The Power of Numbers

Centrioles perform the dual functions of organizing both centrosomes and cilia. The biogenesis of nascent centrioles is an essential cellular event that is tightly coupled to the cell cycle so that each cell contains only two or four centrioles at any given point in the cell cycle. The assembly of centrioles and their analogs, basal bodies, is well characterized at the ultrastructural level whereby structural modules are built into a functional organelle. Genetic studies in model organisms combined with proteomic, bioinformatic and identifying ciliary disease gene orthologs have revealed a wealth of molecules requiring further analysis to determine their roles in centriole duplication, assembly and function. Nonetheless, at this stage, our understanding of how molecular components interact to build new centrioles and basal bodies is limited. The ciliates, Tetrahymena and Paramecium , historically have been the subject of cytological and genetic study of basal bodies. Recent advances in the ciliate genetic and molecular toolkit have placed these model organisms in a favorable position to study the molecular mechanisms of centriole and basal body assembly.     

Destruction of maternal centrioles during fertilization of the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)

In brown algal fertilization, a pair of centrioles is derived from the male gamete, irrespective of the sexual reproduction pattern, i.e., isogamy, anisogamy, or oogamy. In this study, the manner in which the maternal centriole structure is destroyed in early zygotes of the isogamous brown alga Scytosiphon lomentaria was examined by electron microscopy. At fertilization, the zygote had two pairs of centrioles (flagellar basal bodies) derived from motile male and female gametes, and there was no morphological difference between the two pairs. The flagellar basal plate and the axonemal microtubules were still connected with the distal end of centrioles. Ultrastructural observations showed that the integrity of maternal-derived centrioles began to degenerate even in the 1-h-old zygote. At that time, the cylinder of triplet microtubules of the maternal centrioles became shorter from the distal end, and a section passing through the centrioles indicated that a part of the nine triplets of microtubules changed into doublet or singlet microtubules by degeneration of B and/or C tubules. In 2-h-old zygote, there was no trace of maternal centrioles ultrastructurally, and only the paternal centrioles remained. Further, reduction of centrin accompanying destruction of the maternal centrioles was examined in immunofluorescence microscopy. Centrin localized at the paternal and the maternal centrioles had the same fluorescence intensity in the early zygotes. At 4-6 h after fertilization, two spots indicating centrin localization showed different fluorescence intensity. Later, the weaker spot disappeared completely. These results showed that there is a difference in time between the destruction of the centriolar cylinders and the reduction of centrin molecules around them.     

The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.     

Evidence for a functional role of RNA in centrioles.

Basal bodies, purified from Chlamydomonas and Tetrahymena, were exposed to various enzymatic treatments and then assayed for their ability to nucleate aster formation upon injection into eggs of Xenopus laevis. Untreated basal bodies injected into frog eggs act as centrioles and induce the formation of asters. The aster-inducing activity of basal bodies was eliminated by treatment with proteolytic enzymes and ribonucleases. Aster-inducing activity was not affected by DNAse and a number of other enzymes. The effect of proteolytic digestion on aster-inducing activity appeared to be directly correlated with the degree of structural damage to the basal body. Low concentrations of pancreatic ribonuclease A, ribonuclease T1, and S1 nuclease also completely abolished aster-inducing activity, although these enzymes had no effect on basal body structure. Ribonuclease-treated basal bodies remained capable of supporting microtubule elongation in vitro. Preliminary evidence indicates that basal bodies from Chlamydomonas and Tetrahymena contain about 5 x 10(-16) g of RNA which co-band with basal bodies and aster-inducing activity by equilibrium density gradient sedimentation. We conclude first, that centrioles contain RNA which is required for initiation of aster formation, and second, that the centriole activity or ability to assemble a mitotic aster is separable from the basal body activity, or ability to serve directly as a template for microtubule growth.     

Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.     

Basal-body-associated macromolecules: a continuing debate

Controversy over the possibility that centrioles/basal bodies contain nucleic acids has overshadowed results demonstrating other macromolecules in the lumen of these organelles. Glycogen particles, which are known to be present within the lumen of the centriole/basal body of sperm cells, have now been found in basal bodies of protists belonging to three different groups. Here, we extend the debate on a role for RNA in basal body/centriole function and speculate on the origin and the function of centriolar glycogen.     

CENTRIOLE REPLICATION : A Study of Spermatogenesis in the Snail Viviparus

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO₄ and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mµ in diameter and 330 mµ long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.     

Centrioles want to move out and make cilia

Cilia formation in mammalian cells requires basal bodies that are either derived from centrioles that transition from their cytoplasmic role in centrosome organization or that form en masse in multiciliated cells. Several recent studies have begun to uncover the links between centriole duplication and their transformation to basal bodies.     

ULTRASTRUCTURE OF THE ZOOSPORES AND VEGETATIVE CELLS OF TETRAEDRON AND CHLOROTETRAEDRON (CHLOROPHYCEAE)1

The flagellar apparatus of the zoospores of Tetraedron bitridens Beck-Mannagetta and Chlorotetraedron polymorphum MacEntee, Bold et Archibald includes directly opposed basal bodies, a distal fiber that is elaborated into a ribbed structure to which the continuous striated microtubule-associated component (SMAC) is connected, and partial caps over the proximal end of each basal body. The angle between basal bodies ranges from approximately 25° to 150°. Basal bodies at wider angles are interconnected via their cores. A septum is present in the B-tubule of one basal body triplet in C. polymorphum. Both organisms have four microtubular rootlets arranged in a cruciate pattern. The two X-membered rootlets in a single cell have dissimilar numbers of microtubules. In C. polymorphum there are 5 and 6 microtubules in a 4/1 and 5/1 arrangement. 3/1 and 4/1 rootlets are present in T. bitridens. Zoospores of T. bitridens have a fuzzy coat whereas those of C. polymorphum are naked. Pyrenoids in both species are covered by a continuous starch sheath. Vegetative, interphase cells of C. polymorphum have two centrioles connected by a fiber that are located in depressions in the nuclear envelope. We propose that these two genera may be closely related to Neochloris, and that the coenobial genera Hydrodictyon, Pediastrum and Sorastrum are derived from a Tetraedron-like alga.     

Pericentriolar processes of photoreceptor cell basal bodies in the mammalian retina

Pericentriolar processes (arm-like fibers) of the migrating centrioles (diplosome) in differentiating retinal photoreceptor cells were examined in six mammalian species (hamster, vole, rat, rabbit, ferret, cat). These processes emanate in a radial fashion from one end of the centrioles comprising the photoreceptor diplosome. The pericentriolar processes of the basal body are first observed as the diplosome migrates toward the apical plasmalemma, suggesting that centrioles are committed early-on to developing such processes. One pericentriolar process arises from each set of microtubular triplets comprising the centriole and are apicoexternally oriented at an angle of between 30 and 60 degrees with the centriolar axis. Prior to the arrival of one of the centrioles at the apical plasmalemma these processes connect with an electron-dense portion of a centriole-associated vacuole. The diplosome migrates to the apical plasmalemma where one centriole (the presumptive basal body) orients perpendicularly to the apical plasmalemma. The centriole-associated vacuole appears to fuse with the plasmalemma. The pericentriolar processes appear to attach to this fusion site on the plasmalemma which is a region of the membrane characterized by increased electron density (the basilar plate). Invagination of the apical membrane, which occurs at this same site, is accompanied by a lengthening of the microtubules forming a cilium and is observed as an outpouching of the plasmalemma within the aforementioned invagination. The associated vacuole apparently becomes continuous with the apical plasmalemma. These pericentriolar processes appear to be functionally involved in ciliogenesis and offer structural stability between the basal body, the plasmalemma and indirectly the cilium.     

Ultrastructure of spermiogenesis in the gastropod Calliotropis glyptus Watson (Prosobranchia: Trochidae)with special reference to the embedded acrosome

Testicular spermatozoa and sperm development in the archaeogastropod Calliotropis glyptus Watson (Trochoidae: Trochidae) are examined using transmission electron microscopy and formalin-fixed tissues. During spermiogenesis, the acrosome, formed evidently through fusion of Golgi-derived proacrosomal vesicles, becomes deeply embedded in the condensing spermatid nucleus. Two centrioles (proximal and distal), both showing triplet microtubular substructure, are present in spermatids—the distal centriole giving rise to the sperm tail and its associated rootlet. During formation of the basal invagination in the spermatid nucleus, centrioles, and rootlet move towards the nucleus and come to lie totally within the basal invagination. Mitochondria are initially positioned near the base of the nucleus but subsequently become laterally displaced. Morphology of the mature spermatozoon is modified from that of the classic primitive or ect-aquasperm type by having 1) the acrosome embedded in the nucleus (the only known example within the Mollusca), 2) a deep basai invagination in the nucleus containing proximal and distal centrioles and an enveloping matrix (derived from the rootlet), 3) laterally displaced periaxonemal mitochondria, and 4) a tail extending from the basal invagination of the nucleus. Implantation of the acrosomal complex and centrioles within imaginations of the nucleus and lateral displacement of mitochondria effectively minimize the length of the sperm head and midpiece. Such modifications may be associated with motility demands, but this remains to be established. The unusual features of C. glyptus spermatozoa, though easily derivable from ‘typical’ trochoid sperm architecture, may prove useful in delineating the genus Calliotropis or tracing its relationship to other genera within the trochid subfamily Margaritinae.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The first indication of differentiation of the Jensen's ring has been detected in an early stage of spermiogenesis of Felis catus Linné when the pair of centrioles takes up a position immediately beneath the plasma membrane. The chromatoid bodies appear in the early spermatid cytoplasm through the nuclear pore complex. In a more advanced stage, such bodies have been found in association with the striated columns, the distal centriole or the proximal part of flagellum and the Jensen's ring. As the spermiogenesis proceeds, the bodies have decreased their size and density, and finally disappear in mature spermatozoa. The chromatoid bodies seem, therefore, to share with the centriole the capacity to form the connecting piece. As a consequence of disorganization of triplet microtubules of the centriole, a noticeable material appears in the center of lumen of the centriole to be identifiable as a distinct precursor of the central pair of axonemal complex. Microtubules are first developed as the sheath of principal piece of the sperm flagellum, originating from the plasma membrane surrounding the axonemal complex.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE V. BLEPHAROPLAST DEVELOPMENT IN MARCHANTIA POLYMORPHA

Coaxial centrioles and a microtubule organizing center (MTOC) constitute each centrosome in spermatid mother cells of Marchantia polymorpha. During cell division the centrosome separates at its midregion and the two centrioles undergo a planar rotation that brings them to lie somewhat staggered and nearly parallel with their proximal ends embedded in osmiophilic granular material similar in appearance to that of the MTOC. Microtubules of the multilayered structure (MLS) arise in this material below the posterior centriole and parallel to its long axis. The rotation of centrioles and the initiation of S₁ tubules below the posterior centriole determine polarity of the incipient blepharoplast. Lower MLS strata are formed under the anterior centriole by the compaction of granular, osmiophilic matrix. Formation and growth of S₂ vertical lamellae occur at the left front edge of the MLS in association with MTOC-like matrix localized near the cell membrane. The MLS enlarges to about 0.4 μm wide by 0.6 μm long and is ovoid in outline except for a short distal projection underlying the posterior centriole. Subsequently the lamellae are transformed into homogenous, osmiophilic matrix that contributes directly to the expansion of all MLS strata including microtubules. The stratum of lamellae is interpreted as a planar MTOC subject to morphogenetic control. Each of the four strata grows proximally while the tapering distal projection lengthens beneath the posterior basal body. Dense matrix above the MLS, apparently elaborated by the S₂ layer, is organized into cartwheel and triplet components of the basal bodies’ proximal extensions. Organization of triplet tubules proceeds from proximal to distal toward preexisting triplets. Osmiophilic matrix contributes to the formation of microtubule keels and osmiophilic crests and may serve as a cementing material that stabilizes the spatial relationships of blepharoplast components. After full expansion of the MLS’ lower strata, the S₂ layer is reorganized into lamellae. Flagellar growth in Marchantia is postulated to involve a process whereby subunits or their precursors are elaborated by the MLS, translocated to the distal end of the flagellum and incorporated into the axonemal tubules. When MLS microtubules elongate to form a long, narrow band, the distal half of the S₂ layer is again in the osmiophilic matrix state.