scholarly journals The Vfl1 Protein in Chlamydomonas Localizes in a Rotationally Asymmetric Pattern at the Distal Ends of the Basal Bodies

2001 ◽  
Vol 153 (1) ◽  
pp. 63-74 ◽  
Author(s):  
Carolyn D. Silflow ◽  
Matthew LaVoie ◽  
Lai-Wa Tam ◽  
Susan Tousey ◽  
Mark Sanders ◽  
...  
Keyword(s):  
Basal Body ◽  
Rotational Symmetry ◽  
Coiled Coil ◽  
Mutant Phenotype ◽  
Basal Bodies ◽  
Repeat Sequences ◽  
Coiled Coil Domain ◽  
Cell Biol ◽  
Asymmetric Pattern ◽  
Rotational Orientation

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180° rotational symmetry, such that the flagella beat with the effective strokes in opposite directions (Hoops, H.J., and G.B. Witman. 1983. J. Cell Biol. 97:902–908). The vfl1 mutation results in variable numbers and positioning of flagella and basal bodies (Adams, G.M.W., R.L. Wright, and J.W. Jarvik. 1985. J. Cell Biol. 100:955–964). Using a tagged allele, we cloned the VFL1 gene that encodes a protein of 128 kD with five leucine-rich repeat sequences near the NH2 terminus and a large α-helical–coiled coil domain at the COOH terminus. An epitope-tagged gene construct rescued the mutant phenotype and expressed a tagged protein (Vfl1p) that copurified with basal body flagellar apparatuses. Immunofluorescence experiments showed that Vfl1p localized with basal bodies and probasal bodies. Immunogold labeling localized Vfl1p inside the lumen of the basal body at the distal end. Distribution of gold particles was rotationally asymmetric, with most particles located near the doublet microtubules that face the opposite basal body. The mutant phenotype, together with the localization results, suggest that Vfl1p plays a role in establishing the correct rotational orientation of basal bodies. Vfl1p is the first reported molecular marker of the rotational asymmetry inherent to basal bodies.

scholarly journals Bld10p, a novel protein essential for basal body assembly in Chlamydomonas

2004 ◽  
Vol 165 (5) ◽  
pp. 663-671 ◽  
Author(s):  
Kumi Matsuura ◽  
Paul A. Lefebvre ◽  
Ritsu Kamiya ◽  
Masafumi Hirono
Keyword(s):  
Basal Body ◽  
Cell Biology ◽  
Early Stage ◽  
Rotational Symmetry ◽  
Coiled Coil ◽  
Electron Microscopic Observation ◽  
Basal Bodies ◽  
Mitotic Spindles ◽  
Electron Microscopic ◽  
Cytoplasmic Microtubules

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.

scholarly journals Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling

2012 ◽  
Vol 197 (2) ◽  
pp. 313-325 ◽  
Author(s):  
Camille Enjolras ◽  
Joëlle Thomas ◽  
Brigitte Chhin ◽  
Elisabeth Cortier ◽  
Jean-Luc Duteyrat ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Basal Body ◽  
Coiled Coil ◽  
Cell Types ◽  
Sensory Transduction ◽  
Basal Bodies ◽  
Neuronal Cilia ◽  
Complex Process ◽  
Specific Proteins ◽  
And Function

Centriole-to–basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby1/1 flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.

scholarly journals Lrrcc1 and Ccdc61 are conserved effectors of multiciliated cell function

2021 ◽  
Author(s):  
Aude Nommick ◽  
Camille Boutin ◽  
Olivier Rosnet ◽  
Elsa Bazellières ◽  
Virginie Thomé ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Cell Function ◽  
Coiled Coil ◽  
Fluid Flows ◽  
Basal Bodies ◽  
Animal Evolution ◽  
Ciliary Beating ◽  
Coiled Coil Domain ◽  
Multiciliated Cells ◽  
And Function

AbstractCiliated epithelia perform a variety of essential functions across animal evolution, ranging from locomotion of marine organisms to mucociliary clearance of airways in mammals. These epithelia are composed of multiciliated cells (MCCs) harbouring myriads of motile cilia, which rest on modified centrioles called basal bodies (BBs), and beat coordinately to generate directed fluid flows. Thus, BB biogenesis and organization is central to MCC function. In basal eukaryotes, the coiled-coil domain proteins Lrrcc1 and Ccdc61 were shown to be required for proper BB construction and function. Here, we used the Xenopus embryonic ciliated epidermis to characterize Lrrcc1 and Ccdc61 in vertebrate MCCs. We found that they both encode BB components, with a prominent association to striated rootlets. Knocking down either gene caused defects in BB docking, spacing, and polarization. Moreover, their depletion impaired the apical cytoskeleton, and altered ciliary beating. Consequently, cilia-powered fluid flow was greatly reduced in morphant tadpoles, which displayed enhanced mortality when exposed to pathogenic bacteria. This work illustrates how integration across organizational scales make elementary BB components essential for the emergence of the physiological function of ciliated epithelia.

Ultrastructure and development of the flagellar apparatus and flagellar motion in the colonial graeen alga Astrephomene gubernaculifera

1983 ◽  
Vol 63 (1) ◽  
pp. 21-41
Author(s):  
H.J. Hoops ◽  
G.L. Floyd
Keyword(s):  
Basal Body ◽  
Rotational Symmetry ◽  
Amorphous Material ◽  
Flagellar Apparatus ◽  
The Other ◽  
Basal Bodies ◽  
Mature Cell ◽  
Embryonic Cleavage ◽  
Development Proceeds ◽  
Concurrent Events

Immediately following embryonic cleavage, the cells of Astrephomene have four equal-sized basal bodies, two of which are connected by a striated distal fibre and two striated proximal fibres. The four microtubular rootlets, which alternate between having 3/1 and 2 members, are arranged cruciately. The two basal bodies that are connected by the striated fibres then extend into flagella, while the two accessory basal bodies are now markedly shorter. At this stage the flagellar apparatus has 180 degrees rotational symmetry and is very similar to the flagellar apparatus of the unicellular Chlamydomonas and related algae. Development proceeds with a number of concurrent events. The basal bodies begin to separate at their proximal ends and become nearly parallel. Each striated proximal fibre detaches at one end from one of the basal bodies. Each half of the flagellar apparatus, which consists of a flagellum and attached basal body, an accessory basal body, two rootlets and a striated fibre (formerly one of the proximal striated fibres), rotates about 90 degrees, the two halves rotating in opposite directions. An electron-dense strut forms near one two-membered rootlet and grows past both basal bodies. During this time a fine, fibrous component appears between newly developed spade-like structures and associated amorphous material connected to each basal body. The basal bodies continue to separate as the distal fibre stretches and finally detaches from one of them. These processes result in the loss of the 180 degree rotational symmetry present in previous stages. Although the flagella continue to separate, there is no further reorganization of the components of the flagellar apparatus. In the mature cell of Astrephomene, the two flagella are inserted separately and are parallel. The four microtubular rootlets are no longer arranged cruciately. Three of the rootlets are nearly parallel, while the fourth is approximately perpendicular to the other three. A straited fibre connects each basal body to the underside of the strut. These fibres run in the direction of the effective stroke of the flagella and might be important either in anchoring the basal bodies or in the initiation of flagellar motion. Unlike the case in the unicellular Chlamydomonas, the two flagella beat in the same direction and in parallel planes. The flagella of a given cell may or may not beat in synchrony. The combination of this type of flagellar motion and the parallel, separate flagella appears to be suited to the motion of this colonial organism.

The ultrastructure of Ulothrix mucosa. II. The flagellar apparatus of the zoospore

1986 ◽  
Vol 64 (1) ◽  
pp. 166-176 ◽  
Author(s):  
G. M. Lokhorst ◽  
W. Star
Keyword(s):  
Basal Body ◽  
Spatial Configuration ◽  
Rotational Symmetry ◽  
Flagellar Apparatus ◽  
Taxonomic Status ◽  
Proximal Region ◽  
Basal Bodies ◽  
Serial Sectioning ◽  
Counterclockwise Direction ◽  
Microtubular Roots

The actual spatial configuration of the flagellar apparatus of the quadriflagellate zoospore of Ulothrix mucosa Thuret has been reconstructed by serial sectioning analysis. This apparatus shows an architecture quite similar to that found in related Ulvophyceae. Common characteristics are the differently leveled basal body pairs; the 180° rotational symmetry of the flagellar apparatus; the proximal overlap of the upper basal bodies which are displaced with respect to each other in the counterclockwise direction; terminal caps; four cruciately arranged microtubular roots (R2, R4); a distinctly striated distal connecting fibre that interconnects the upper basal bodies; and striated bands (SB1) that join the R4s to the lower basal bodies. Specific features are the arrangement of the R4 in a three over one configuration when entering the proximal region of the flagellar apparatus; the differently shaped proximal sheaths and their association with a proximal sheath connecting band; the presence of two system II fibres (rhizoplasts) which arise from the lower basal body pair; the striated bands (SB2) that connect the R2s to the lower basal bodies; the distinct striation of the system I fibre, which is not only intimately associated with the R2, but also with the R4 (not earlier reported for an ulvophycean alga); and, finally, the relevant displacement of the lower basal body pair in a counterclockwise direction of approximately half a basal body diameter. In light of these findings the taxonomic status of the Ulotrichales as well as of the Ulvophyceae is discussed.

scholarly journals Genetic Evidence for Interaction between η- and β-Tubulins

2004 ◽  
Vol 3 (1) ◽  
pp. 212-220 ◽  
Author(s):  
F. Ruiz ◽  
P. Dupuis-Williams ◽  
C. Klotz ◽  
F. Forquignon ◽  
M. Bergdoll ◽  
...  
Keyword(s):  
Basal Body ◽  
Mutant Phenotype ◽  
Basal Bodies ◽  
Paramecium Tetraurelia ◽  
Nonpermissive Temperature ◽  
Genetic Approach ◽  
Gene Coding ◽  
End Capping ◽  
Extragenic Suppressors ◽  
Β Tubulin

ABSTRACT The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, α to ε, characterized in P. tetraurelia. In order to analyze η-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the β-PT2 gene coding for a β-tubulin, while the mutation nocr 1 conferring nocodazole resistance and localized in another β-tubulin gene, β-PT3, was shown to enhance the mutant phenotype. The interaction between η-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that η-tubulin, like γ- and δ-tubulins, might be a microtubule minus-end capping molecule. The likely function of η-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.

scholarly journals Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet

2002 ◽  
Vol 159 (3) ◽  
pp. 431-440 ◽  
Author(s):  
Jun Yang ◽  
Xiaoqing Liu ◽  
Guohua Yue ◽  
Michael Adamian ◽  
Oleg Bulgakov ◽  
...  
Keyword(s):  
Basal Body ◽  
Structural Component ◽  
Coiled Coil ◽  
Basal Bodies ◽  
Tail Domain ◽  
Kinesin Light Chain ◽  
Thick Filaments ◽  
Head Domain ◽  
Retinal Photoreceptors

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Despite being the largest cytoskeleton, its structural composition has remained unknown. Here, we report a novel 220-kD protein, designated rootletin, found in the rootlets of ciliated cells. Recombinant rootletin forms detergent-insoluble filaments radiating from the centrioles and resembling rootlets found in vivo. An mAb widely used as a marker for vertebrate rootlets recognizes an epitope in rootletin. Rootletin has a globular head domain and a tail domain consisting of extended coiled-coil structures. Rootletin forms parallel in register homodimers and elongated higher order polymers mediated by the tail domain alone. The head domain may be required for targeting to the basal body and binding to a kinesin light chain. In retinal photoreceptors where rootlets appear particularly robust, rootlets extend from the basal bodies to the synaptic terminals and anchor ER membranes along their length. Our data indicate that rootlets are composed of homopolymeric rootletin protofilaments bundled into variably shaped thick filaments. Thus, rootletin is the long-sought structural component of the ciliary rootlet.

scholarly journals Pea2 protein of yeast is localized to sites of polarized growth and is required for efficient mating and bipolar budding.

1996 ◽  
Vol 135 (3) ◽  
pp. 725-739 ◽  
Author(s):  
N Valtz ◽  
I Herskowitz
Keyword(s):  
Coiled Coil ◽  
Cell Types ◽  
Cell Polarization ◽  
Polarized Growth ◽  
Coiled Coil Domain ◽  
Cell Biol ◽  
Two Phases ◽  
Diploid Cells ◽  
Novel Protein

Saccharomyces cerevisiae exhibits polarized growth during two phases of its life cycle, budding and mating. The site for polarization during vegetative growth is determined genetically: a and alpha haploid cells exhibit an axial budding pattern, and a/alpha diploid cells exhibit a bipolar pattern. During mating, each cell polarizes towards its partner to ensure efficient mating. SPA2 is required for the bipolar budding pattern (Snyder. M 1989. J. Cell Biol. 108:1419-1429; Zahner, J.A., H.A. Harkins, and J.R. Pringle. 1996. Mol. Cell. Biol. 16:1857-1870) and polarization during mating (Snyder, M., S. Gehrung, and B.D. Page. 1991. J. Cell Biol. 114: 515-532). We previously identified mutants defective in PEA2 and SPA2 which alter cell polarization in the presence of mating pheromone in a similar manner (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics, 136:1287-1297). Here we report the further characterization of these mutants. We have found that PEA2 is also required for the bipolar budding pattern and that it encodes a novel protein with a predicted coiled-coil domain. Pea2p is expressed in all cell types and is localized to sites of polarized growth in budding and mating cells in a pattern similar to Spa2p, Pea2p and Spa2p exhibit interdependent localization: Spa2p is produced in pea2 mutants but fails to localize properly; Pea2p is not stably produced in spa2 mutants. These results suggest that Pea2p and Spa2p function together as a complex to generate the bipolar budding pattern and to guarantee proper polarization during mating.

Ciliary coordination in newt lung cells requires microtubule-based linkages between basal bodies: A correlative light and EM study

1992 ◽  
Vol 50 (1) ◽  
pp. 570-571
Author(s):  
Robert Hard ◽  
Gerald Rupp ◽  
Matthew L. Withiam-Leitch ◽  
Lisa Cardamone
Keyword(s):  
Basal Body ◽  
Untreated Control ◽  
Beat Frequency ◽  
Primary Cultures ◽  
Living Cells ◽  
Power Stroke ◽  
Ultrastructural Changes ◽  
Lung Cells ◽  
Basal Bodies ◽  
Ciliated Cells

In a coordinated field of beating cilia, the direction of the power stroke is correlated with the orientation of basal body appendages, called basal feet. In newt lung ciliated cells, adjacent basal feet are interconnected by cold-stable microtubules (basal MTs). In the present study, we investigate the hypothesis that these basal MTs stabilize ciliary distribution and alignment. To accomplish this, newt lung primary cultures were treated with the microtubule disrupting agent, Colcemid. In newt lung cultures, cilia normally disperse in a characteristic fashion as the mucociliary epithelium migrates from the tissue explant. Four arbitrary, but progressive stages of dispersion were defined and used to monitor this redistribution process. Ciliaiy beat frequency, coordination, and dispersion were assessed for 91 hrs in untreated (control) and treated cultures. When compared to controls, cilia dispersed more rapidly and ciliary coordination decreased markedly in cultures treated with Colcemid (2 mM). Correlative LM/EM was used to assess whether these effects of Colcemid were coupled to ultrastructural changes. Living cells were defined as having coordinated or uncoordinated cilia and then were processed for transmission EM.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum

2019 ◽  
Vol 476 (21) ◽  
pp. 3241-3260
Author(s):  
Sindhu Wisesa ◽  
Yasunori Yamamoto ◽  
Toshiaki Sakisaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Transmembrane Domains ◽  
Domain Family ◽  
Coiled Coil Domain ◽  
U2os Cells ◽  
Er Membrane

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

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