In vitro reassembly of basal body components

1984 ◽  
Vol 66 (1) ◽  
pp. 147-154
Author(s):  
R.H. Gavin
Keyword(s):  
Basal Body ◽  
Basal Bodies ◽  
Body Components

Singlet microtubules and structures that have the morphology and dimensions of the basal body cartwheel complex self-assemble in extracts derived from Tetrahymena basal bodies.

In vitro effects of taxol on ciliogenesis in quail oviduct

1989 ◽  
Vol 92 (1) ◽  
pp. 9-20 ◽  
Author(s):  
E. Boisvieux-Ulrich ◽  
M.C. Laine ◽  
D. Sandoz
Keyword(s):  
Basal Body ◽  
In Situ Polymerization ◽  
Neck Region ◽  
Apical Part ◽  
Transitional Region ◽  
Basal Bodies ◽  
Apical Surface ◽  
In Vitro Effects

When induced by in vivo oestrogen stimulation, ciliogenesis continues in culture in vitro of quail oviduct implants. Ultrastructure of ciliogenic cells was compared after culture for 24 or 48 h in the presence or absence of 10(−5) M-taxol. Taxol, which promotes polymerization and stabilization of microtubules, disturbed ciliogenesis, but formation of basal bodies was unaffected by the drug. Conversely, their migration towards the apical surface seemed to be slowed down or blocked and axonemal doublets polymerized onto the distal end of cytoplasmic basal bodies. They elongated and often constituted a more or less complete axoneme, extending between organelles in various orientations. These axonemes, often abnormal, were not surrounded by a membrane, with the exception of the transitional or neck region between the basal body and axoneme. The formation of membrane in this area resulted from the binding of some vesicles to the anchoring fibres of the basal body. They fused in various numbers, occasionally forming a ring, at the site of the transitional region, and exhibited the characteristics of the ciliary necklace. The association of basal bodies with vesicles or with the plasma membrane appeared to be a necessary signal for in situ polymerization of axonemal doublets. In addition, taxol induced polymerization of numerous microtubules in the cytoplasm, especially in the apical part of the cell and in the Golgi area. This network of microtubules may prevent basal body migration.

scholarly journals Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

2021 ◽  
pp. mbc.E20-11-0717
Author(s):  
Jaime V.K. Hibbard ◽  
Neftali Vazquez ◽  
Rohit Satija ◽  
John B. Wallingford
Keyword(s):  
Protein Dynamics ◽  
Basal Body ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Basal Bodies ◽  
Multiciliated Cells ◽  
Capture Mechanism ◽  
Body Components ◽  
Ciliary Signaling

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

Gene action in development

1970 ◽  
Vol 176 (1044) ◽  
pp. 347-366 ◽  
Keyword(s):  
Basal Body ◽  
Spatial Relations ◽  
Building Blocks ◽  
Control Synthesis ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Negative Results ◽  
Molecular Building Blocks

This paper deals with processes occurring in the assembly of genic products into cell organelles and structures, especially with processes determining the initiation, location, orientation and number of cortical structures in the ciliated protozoan, Paramecium aurelia . The examples analysed experimentally are: ciliary basal bodies and their associated structures, the repeating unit territory of cortical structure, the pattern of path directions of the rows of basal bodies and unit territories, the distribution in the cortex of two different kinds of unit territories, and the ingestatory apparatus (vestibule and gullet). Among the processes that appear to occur are homonucleation, heteronucleation, and allosteric transitions, as found by others in the in vitro assembly of bacterial flagella, bacterial viruses, and the parts of ciliary microtubules and their appendages. In Paramecium , new basal bodies can arise only in one position and orientation, close and at right angles to an existing basal body at a specified spot. Unit territories reproduce by forming additional parts and subdividing. The information for the positioning and orientation of the developing parts of the unit territory is located within the unit itself and, when experimentally altered, reproduces in the altered orientation which cannot be corrected by genic action. This hereditary aspect of development is determined by an unbroken chain of self-reproducing arrangements of cortical parts. Search for DNA in the cortex gave negative results. Analysis of the hereditary determination of initiation, location, and orientation of the gullet gave results similar to those on the basal body and unit territory. Analysis of the hereditary determination of the path of the rows of unit territories and basal bodies and of the distribution of the two kinds of unit territories showed developmental and genetic control by spatial relations to the vestibule-gullet. Thus, for all corticaltra its examined, development is hereditarily determined by existing and self-reproducing cortical arrangements: the genes (or DNA) doubtless control synthesis of the molecular building blocks, but not their site of assembly or the position, orientation and number of assemblies. The flatworm Stenostomum also shows hereditary developmental control by self-perpetuating structural arrangements, two variations from normal (two kinds of doublet worms) reproducing true to type during asexual reproduction. Whether similar processes occur in the inheritance of the developmentally decisive organization of the amphibian egg is still an open question The processes described in this paper constitute but one of many options available for hereditary control of development.

scholarly journals Basal Body Components Exhibit Differential Protein Dynamics during Nascent Basal Body Assembly

2009 ◽  
Vol 20 (3) ◽  
pp. 904-914 ◽  
Author(s):  
Chad G. Pearson ◽  
Thomas H. Giddings ◽  
Mark Winey
Keyword(s):  
Basal Body ◽  
Tetrahymena Thermophila ◽  
Light And Electron Microscopy ◽  
Protein Assembly ◽  
Basal Bodies ◽  
Body Formation ◽  
Unique Protein ◽  
Body Components ◽  
And Function ◽  
Stable Populations

Basal bodies organize cilia that are responsible for both mechanical beating and sensation. Nascent basal body assembly follows a series of well characterized morphological events; however, the proteins and their assembly dynamics for new basal body formation and function are not well understood. High-resolution light and electron microscopy studies were performed in Tetrahymena thermophila to determine how proteins assemble into the structure. We identify unique dynamics at basal bodies for each of the four proteins analyzed (α-tubulin, Spag6, centrin, and Sas6a). α-Tubulin incorporates only during new basal body assembly, Spag6 continuously exchanges at basal bodies, and centrin and Sas6a exhibit both of these patterns. Centrin loads and exchanges at the basal body distal end and stably incorporates during new basal body assembly at the nascent site of assembly and the microtubule cylinder. Conversely, both dynamic and stable populations of Sas6a are found only at a single site, the cartwheel. The bimodal dynamics found for centrin and Sas6a reveal unique protein assembly mechanisms at basal bodies that may reflect novel functions for these important basal body and centriolar proteins.

scholarly journals Protein turnover dynamics suggest a diffusion to capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

2020 ◽  
Author(s):  
Jaime V.K. Hibbard ◽  
Neftali Vazquez ◽  
Rohit Satija ◽  
John B. Wallingford
Keyword(s):  
Protein Dynamics ◽  
Basal Body ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Basal Bodies ◽  
Multiciliated Cells ◽  
Capture Mechanism ◽  
Body Components

ABSTRACTIntraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body, where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins, using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT sub-complexes displayed shared dynamics, and these dynamics were not altered during cilia regeneration as compared to homeostasis. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis.

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.

scholarly journals The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 635-641 ◽  
Author(s):  
J L Salisbury ◽  
A T Baron ◽  
M A Sanders
Keyword(s):  
Nuclear Envelope ◽  
Polyclonal Antibodies ◽  
Flagellar Apparatus ◽  
Immunogold Labeling ◽  
Basal Bodies ◽  
Flagellar Roots ◽  
Cell Biol ◽  
Immunofluorescence Studies ◽  
Descending Fibers

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

scholarly journals Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

2015 ◽  
Vol 197 (11) ◽  
pp. 1921-1930 ◽  
Author(s):  
Jennifer Tsang ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Basal Body ◽  
Protein Interactions ◽  
Transcript Levels ◽  
Content Type ◽  
Genes Encoding ◽  
H Pylori ◽  
Flagellar Biogenesis ◽  
Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

Structural Analysis of Basal Bodies of The Isolated Oral Apparatus of Tetrahymena Pyriformis

1970 ◽  
Vol 6 (3) ◽  
pp. 679-700
Author(s):  
J. WOLFE
Keyword(s):  
Structural Analysis ◽  
Cell Membrane ◽  
Basal Body ◽  
Structural Integrity ◽  
Tetrahymena Pyriformis ◽  
Basal Plate ◽  
Basal Bodies ◽  
The Third ◽  
Ionic Detergent

The oral apparatus of Tetrahymena pyriformis was isolated using a non-ionic detergent to disrupt the cell membrane. The mouth consists largely of basal bodies and microfilaments. Each basal body is attached to the mouth by a basal plate which is integrated into the meshwork of microfilaments that confers upon the oral apparatus its structural integrity. Each basal body is composed of 9 triplet microtubules. Two of the 3 tubules, subfibres ‘A’ and ‘B’ are composed of filamentous rows of globules with a spacing of 4.5nm. The third tubule, subfibre ‘C’, is only one-third the length of the basal body.

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