scholarly journals Three-dimensional Organization of Basal Bodies from Wild-Type and δ-Tubulin Deletion Strains of Chlamydomonas reinhardtii

2003 ◽  
Vol 14 (7) ◽  
pp. 2999-3012 ◽  
Author(s):  
Eileen T. O'Toole ◽  
Thomas H. Giddings ◽  
J. Richard McIntosh ◽  
Susan K. Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Specimen Preparation ◽  
Basal Bodies ◽  
Wild Type ◽  
Tubulin Isoforms ◽  
Striated Fiber ◽  
And Function

Improved methods of specimen preparation and dual-axis electron tomography have been used to study the structure and organization of basal bodies in the unicellular alga Chlamydomonas reinhardtii. Novel structures have been found in both wild type and strains with mutations that affect specific tubulin isoforms. Previous studies have shown that strains lacking δ-tubulin fail to assemble the C-tubule of the basal body. Tomographic reconstructions of basal bodies from the δ-tubulin deletion mutant uni3-1 have confirmed that basal bodies contain mostly doublet microtubules. Our methods now show that the stellate fibers, which are present only in the transition zone of wild-type cells, repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in α-tubulin. tua2-6; uni3-1 cells build both flagella, yet they retain defects in basal body structure and in rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms in Chlamydomonas directly affects the assembly and function of both basal bodies and basal body-associated structures.

Extragenic Bypass Suppressors of Mutations in the Essential Gene BLD2 Promote Assembly of Basal Bodies With Abnormal Microtubules in Chlamydomonas reinhardtii

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 163-181
Author(s):  
Andrea M Preble ◽  
Thomas H Giddings ◽  
Susan K Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Insertional Mutagenesis ◽  
Essential Gene ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Lethal Phenotype ◽  
Flagellar Assembly ◽  
And Function

Abstract bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.

scholarly journals A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation.

1985 ◽  
Vol 101 (5) ◽  
pp. 1903-1912 ◽  
Author(s):  
R L Wright ◽  
J Salisbury ◽  
J W Jarvik
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Nuclear Dna ◽  
Electron Microscopic Observation ◽  
Variable Number ◽  
Immunofluorescent Staining ◽  
Major Protein ◽  
Basal Bodies ◽  
Electron Microscopic ◽  
And Function

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus-basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.

scholarly journals BALD-2: a mutation affecting the formation of doublet and triplet sets of microtubules in Chlamydomonas reinhardtii.

1975 ◽  
Vol 66 (3) ◽  
pp. 480-491 ◽  
Author(s):  
U W Goodenough ◽  
H S StClair
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Transition Region ◽  
Structural Stability ◽  
Mutant Strain ◽  
Basal Body ◽  
Fiber Material ◽  
Basal Bodies ◽  
Critical Properties ◽  
Striated Fiber

The mutant strain bald-2 is unique among "flagellaless" strains of Chlamydomonas reinhardtii isolated to date, in that it possesses a mutant basal body: it is only capable of forming a ring of nine singlet microtubules, 180 nm in diameter, instead of the usual triplet basal body which is 225 nm in diameter. This singlet basal body lacks structural stability and the ability to associate with striated fiber material but retains two critical properties of basal bodies, namely, information specifying the length to which it should elongate and the ability to induce, albeit rarely, a flagellar transition region, a short, singlet-containing axoneme, and a specialized tunnel in the cell wall through which flagella normally emerge. The mutation seems to be specific for B- and C-microtubule synthesis or assembly since all other cytoplasmic sets of microtubules appear normal in numbers, orientation, and stability.

Development of surface pattern during division in Paramecium. I. Mapping of duplication and reorganization of cortical cytoskeletal structures in the wild type

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 191-211 ◽  
Author(s):  
F. Iftode ◽  
J. Cohen ◽  
F. Ruiz ◽  
A.T. Rueda ◽  
L. Chen-Shan ◽  
...  
Keyword(s):  
Basal Body ◽  
Surface Pattern ◽  
Basal Bodies ◽  
Wild Type ◽  
Cell Level ◽  
Local Constraints ◽  
Whole Cell ◽  
Cortical Unit ◽  
Asymmetrical Pattern ◽  
In The Wild

The shape of a Paramecium is determined by the organization of its cortex which constitutes most of the cell cytoskeleton. These structures and networks are organized in relation to the approx. 4000 ciliary basal bodies present at the surface. Each basal body is the centre of a polarized and asymmetrical cortical unit. At the whole-cell level, all units are tandemly arranged in parallel rows and form a defined asymmetrical pattern with dorsoventral and anteroposterior polarities. During division, the cortex is the site of the major morphogenetic processes. In order to analyse how the surface pattern and the shape of the cell are reconstructed at each division, we have used specific immunological and cytological probes to map, in space and time, the reorganization of each of the major cytoskeletal cortical components: basal bodies and microtubules, kinetodesmal fibres, epiplasm and outer lattice. This cytological dissection demonstrates that the surface of the dividing cell is progressively invaded by morphogenetic waves which successively and individually trigger the duplication, assembly or reorganization of each structure and which all spread from the same epicentre (oral apparatus and fission furrow) with the same shape. Furthermore, the response of units to the morphogenetic waves depends on their position on the cell. It thus appears that despite the structural local constraints within units, the development of surface pattern is controlled in an integrated manner by transcellular signals.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells

2004 ◽  
Vol 286 (4) ◽  
pp. L650-L657 ◽  
Author(s):  
Yingjian You ◽  
Tao Huang ◽  
Edward J. Richer ◽  
Jens-Erik Harboe Schmidt ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basal Body ◽  
Ciliated Cell ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Basal Bodies ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Wild Type

Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals Electron Tomography

2002 ◽  
Vol 10 (2) ◽  
pp. 3-5
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Two Dimensions ◽  
Three Dimensions ◽  
Specimen Preparation ◽  
Two Dimensional Image ◽  
Transmission Electron ◽  
Different Depths ◽  
Depth Dimension ◽  
Pass Through

The transmission electron microscope (TEM) was invented in the 1930's, and developments in specimen preparation in the 1950's led to its widespread use as a tool to study structure in biologic systems. Similar in principle to the light microscope, but utilizing a much shorter wavelength for better resolution, the TEM has the image-forming beam pass through the specimen. This results in a two-dimensional image which can be difficult to interpret because features from different depths of the three dimensional specimen are superimposed. Traditionally this was dealt with by cutting sections of plastic-embedded specimens so thin (in the 40 to SO nanometer range) that they effectively had only two dimensions. To allow biologists to examine structures in three dimensions, serial sections are stacked and structures reconstructed. Even though computers have made reconstruction easier, the reality is that resolution in the depth dimension is limited by the section thickness. The technique of electron tomography is emerging as a way to overcome this limitation.

The ultrastructure of the somatic cortex of Pseudomicrothorax dubius: structure and function of the epiplasm in ciliated protozoa

1977 ◽  
Vol 25 (1) ◽  
pp. 367-385
Author(s):  
R.K. Peck
Keyword(s):  
Cell Surface ◽  
Basal Body ◽  
Structure And Function ◽  
Basal Bodies ◽  
Ciliated Protozoa ◽  
Somatic Cortex ◽  
And Function

The ultrastructure of the somatic cortex of the ciliate Pseudomicrothorax dubius is studied with emphasis on the epiplasm layer which lies immediately under the inner alveolar membrane and is continuous with the terminal plates of cortical basal bodies. In addition to a clearly demonstrable cytoskeletal role, the epiplasm appears to function as a comenting substance which integrates numerous cortical fibres and membranes. The kinetodesmal, postciliary and transverse fibre systems which originate at the proximal ends of basal bodies extend toward the cell surface and end at or in the epiplasm. Inner alveolar membranes and trichocyst membranes are attached to the epiplasm. Basal bodies are anchored into the epiplasm via their terminal plates. The epiplasm appears to be morphogenetically important as a matrix into which newly formed basal bodies insert. Electron-opaque arms occur at the terminal plate level of new basal bodies, and these arms fuse with the epiplasm when basal body insertion occurs. The position of trichocysts in the cortex is specified by the epiplasm. Evidence from numerous other ciliates tends to confirm both structural and morphogenetic roles of the epiplasm.

scholarly journals The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization.

1975 ◽  
Vol 65 (1) ◽  
pp. 65-74 ◽  
Author(s):  
R R Gould
Keyword(s):  
Electron Microscopy ◽  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Sodium Dodecyl ◽  
Basal Bodies ◽  
Polyethylene Glycol 400 ◽  
Thin Sections ◽  
Particulate Contamination ◽  
Whole Mount ◽  
Carbon Coated

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.

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